Alkaline phosphatase composition, method of producing dephosphorylated nucleic acid and method of producing labeled nucleic acid

ABSTRACT

A composition contains an alkaline phosphatase; and a peptide fragment group (A) composed of two or more peptide fragments, wherein each of the two or more peptide fragments consists of 5 to 50 consecutive amino acid residues selected from positions 501 to 578 of the amino acid sequence set forth in SEQ ID NO: 5, wherein a content ratio of the peptide fragment group (A) to the alkaline phosphatase satisfies formula (A): (XA/Y)×100≤4.4000 (A), wherein XA represents a peak area value of the peptide fragment group (A) calculated by an automatic integration method from an extracted ion chromatogram obtained by an LC-MS/MS analysis of the composition, and Y represents a peak area value of the alkaline phosphatase calculated by an automatic integration method from a chromatogram obtained by an LC-UV analysis of the composition.

TECHNICAL FIELD

This disclosure relates to a composition containing an alkalinephosphatase, a method of producing a dephosphorylated nucleic acid byusing the composition and a method of producing a labeled nucleic acidby using the composition.

BACKGROUND

An alkaline phosphatase has a catalyst function that hydrolyzesphosphoric monoesters, and has been widely used in methods of measuringthe amount of biological substances such as proteins and nucleic acids(e.g., the immunostaining method, ELISA, the nucleic acid microarraymethod or the like). For example, in the research field of geneticengineering, for pretreatment of labeling of nucleic acids such as DNAand RNA and prevention of self-ligation of vectors, dephosphorylation ofthe 5′ end and/or the 3′ end of a nucleic acid with an alkalinephosphatase has been performed.

As an industrial production method of an alkaline phosphatase, aproduction method in which bovine small intestine or large intestine ismainly used as a raw material has been widely adopted since the specificactivity of the produced alkaline phosphatase is high. The specificactivity of an alkaline phosphatase is generally evaluated by measuringthe absorbance at 405 nm derived from p-nitrophenol produced whenp-nitrophenylphosphate is decomposed.

The quality of an alkaline phosphatase has been evaluated based on thealkaline phosphatase specific activity. To obtain an alkalinephosphatase having a higher specific activity than that of an alkalinephosphatase derived from bovine intestine, an alkaline phosphatasehaving a high specific activity has been isolated in a purificationprocess or has been produced by using recombinant Escherichia coliobtained by a genetic engineering method.

JP H10-262674 A discloses a method of producing an alkaline phosphatasehaving a high specific activity by using recombinant Escherichia coliinto which an alkaline phosphatase-encoding gene derived from the genusBacillus badius has been introduced. WO 2012/115023 discloses a methodof producing an alkaline phosphatase having a high specific activity andheat resistance by using recombinant Escherichia coli into which analkaline phosphatase-encoding gene derived from the genus Shewanella hasbeen introduced.

A dephosphorylation reagent containing an alkaline phosphatase (e.g., acommercially available alkaline phosphatase product) is a compositioncontaining other components in addition to the alkaline phosphatase. Aquality of a dephosphorylation reagent containing an alkalinephosphatase is evaluated based on the alkaline phosphatase specificactivity.

However, we found that, even if labeled nucleic acids prepared by usingdephosphorylation reagents having almost the same alkaline phosphatasespecific activity (labeled nucleic acids obtained by dephosphorylatingthe 5′ ends and/or the 3′ ends of nucleic acids with thedephosphorylation reagents, and then binding labeling substances to the5′ ends and/or the 3′ ends of the dephosphorylated nucleic acids) areused for a nucleic acid detection method, a great difference in thedetection sensitivity between the labeled nucleic acids may occur in thenucleic acid detection method. In other words, we found that the qualityof a dephosphorylation reagent containing an alkaline phosphatase cannotbe evaluated correctly by using the alkaline phosphatase specificactivity as an index.

Thus, it could be helpful to provide a composition containing analkaline phosphatase and having a high quality, a method of producing adephosphorylated nucleic acid by using the composition and a method ofproducing a labeled nucleic acid by using the composition.

SUMMARY

We found that at least any one of the following impurities can coexistin a dephosphorylation reagent containing an alkaline phosphatase (e.g.,a commercially available alkaline phosphatase product):

a peptide fragment group (A) composed of two or more peptide fragments,wherein each of the two or more peptide fragments consists of 5 to 50consecutive amino acid residues selected from positions 501 to 578 ofthe amino acid sequence set forth in SEQ ID NO: 5 (which corresponds toan amino acid sequence of a bovine-derived alkaline phosphatase);

a peptide fragment group (B) composed of two or more peptide fragments,wherein each of the two or more peptide fragments consists of 13 to 50consecutive amino acid residues selected from positions 501 to 578 ofthe amino acid sequence set forth in SEQ ID NO: 5 and contains positions516 to 528 of the amino acid sequence set forth in SEQ ID NO: 5;

a peptide fragment group (C) composed of two or more peptide fragments,wherein each of the two or more peptide fragments consists of 12 to 50consecutive amino acid residues selected from positions 501 to 578 ofthe amino acid sequence set forth in SEQ ID NO: 5 and contains positions534 to 545 of the amino acid sequence set forth in SEQ ID NO: 5;

a first peptide fragment consisting of the amino acid sequence set forthin SEQ ID NO: 1;

a second peptide fragment consisting of the amino acid sequence setforth in SEQ ID NO: 2;

a third peptide fragment consisting of the amino acid sequence set forthin SEQ ID NO: 3; and

a fourth peptide fragment consisting of the amino acid sequence setforth in SEQ ID NO: 4.

In addition, we found that, by reducing, in a dephosphorylation reagentwhich is used to prepare a labeled nucleic acid (a labeled nucleic acidobtained by dephosphorylating the 5′ end and/or the 3′ end of a nucleicacid with the dephosphorylation reagent, and then binding a labelingsubstance to the 5′ end and/or the 3′ end of the dephosphorylatednucleic acid) for a nucleic acid detection method,

the content of the peptide fragment group (A),

the content of the peptide fragment group (B),

the content of the peptide fragment group (C),

the content of the second peptide fragment (preferably, the content ofthe second peptide fragment, and the content(s) of one, two or threepeptide fragments selected from the first, third and fourth peptidefragments),

the content of the third peptide fragment (preferably, the content ofthe third peptide fragment, and the content(s) of one, two or threepeptide fragments selected from the first, second and fourth peptidefragments), or

the content of the fourth peptide fragment (preferably, the content ofthe fourth peptide fragment, and the content(s) of one, two or threepeptide fragments selected from the first, second and third peptidefragments),

it is possible to improve the detection sensitivity of the labelednucleic acid in the nucleic acid detection method, thus completing thisdisclosure.

We thus provide:

-   [1] A composition containing:

an alkaline phosphatase; and

a peptide fragment group (A) composed of two or more peptide fragments,wherein each of the two or more peptide fragments consists of 5 to 50consecutive amino acid residues selected from positions 501 to 578 ofthe amino acid sequence set forth in SEQ ID NO: 5,

wherein a content ratio of the peptide fragment group (A) to thealkaline phosphatase satisfies formula (A):

(X _(A) /Y)×100≤4.4000   (A),

wherein X_(A) represents a peak area value of the peptide fragment group(A) calculated by an automatic integration method from an extracted ionchromatogram obtained by an LC-MS/MS analysis of the composition, and Yrepresents a peak area value of the alkaline phosphatase calculated byan automatic integration method from a chromatogram obtained by an LC-UVanalysis of the composition.

-   [2] The composition according to [1], wherein the peptide fragment    group (A) contains one, two, three or four peptide fragments    selected from the group consisting of a first peptide fragment    consisting of the amino acid sequence set forth in SEQ ID NO: 1, a    second peptide fragment consisting of the amino acid sequence set    forth in SEQ ID NO: 2, a third peptide fragment consisting of the    amino acid sequence set forth in SEQ ID NO: 3 and a fourth peptide    fragment consisting of the amino acid sequence set forth in SEQ ID    NO: 4.-   [3] The composition according to [2], wherein:

the peptide fragment group (A) contains the first peptide fragment; and

a content ratio of the first peptide fragment to the alkalinephosphatase satisfies formula (1):

(X ₁ /Y)×100≤1.0000   (1),

wherein X₁ represents a peak area value of the first peptide fragmentcalculated by an automatic integration method from an extracted ionchromatogram obtained by an LC-MS/MS analysis of the composition, and Yis the same as defined above.

-   [4] The composition according to [2] or [3], wherein:

the peptide fragment group (A) contains the second peptide fragment; and

a content ratio of the second peptide fragment to the alkalinephosphatase satisfies formula (2):

(X ₂ /Y)×100≤1.6000   (2),

wherein X₂ represents a peak area value of the second peptide fragmentcalculated by an automatic integration method from an extracted ionchromatogram obtained by an LC-MS/MS analysis of the composition, and Yis the same as defined above.

-   [5] The composition according to any one of [2] to [4], wherein:

the peptide fragment group (A) contains the third peptide fragment; and

a content ratio of the third peptide fragment to the alkalinephosphatase satisfies formula (3):

(X ₃ /Y)×100≤0.2000   (3),

wherein X₃ represents a peak area value of the third peptide fragmentcalculated by an automatic integration method from an extracted ionchromatogram obtained by an LC-MS/MS analysis of the composition, and Yis the same as defined above.

-   [6] The composition according to any one of [2] to [5], wherein:

the peptide fragment group (A) contains the fourth peptide fragment; and

a content ratio of the fourth peptide fragment to the alkalinephosphatase satisfies formula (4):

(X ₄ /Y)×100≤0.3500   (4),

wherein X₄ represents a peak area value of the fourth peptide fragmentcalculated by an automatic integration method from an extracted ionchromatogram obtained by an LC-MS/MS analysis of the composition, and Yis the same as defined above.

-   [7] A composition containing:

an alkaline phosphatase; and

a peptide fragment group (B) composed of two or more peptide fragments,wherein each of the two or more peptide fragments consists of 13 to 50consecutive amino acid residues selected from positions 501 to 578 ofthe amino acid sequence set forth in SEQ ID NO: 5 and contains positions516 to 528 of the amino acid sequence set forth in SEQ ID NO: 5,

wherein a content ratio of the peptide fragment group (B) to thealkaline phosphatase satisfies formula (B):

(X _(B) /Y)×100≤3.4000   (B),

wherein X_(B) represents a peak area value of the peptide fragment group(B) calculated by an automatic integration method from an extracted ionchromatogram obtained by an LC-MS/MS analysis of the composition, and Yrepresents a peak area value of the alkaline phosphatase calculated byan automatic integration method from a chromatogram obtained by an LC-UVanalysis of the composition.

-   [8] The composition according to [7], wherein the peptide fragment    group (B) contains one or two peptide fragments selected from the    group consisting of a first peptide fragment consisting of the amino    acid sequence set forth in SEQ ID NO: 1 and a second peptide    fragment consisting of the amino acid sequence set forth in SEQ ID    NO: 2.-   [9] The composition according to [8], wherein:

the peptide fragment group (B) contains the first peptide fragment; and

a content ratio of the first peptide fragment to the alkalinephosphatase satisfies formula (1):

(X ₁ /Y)×100≤1.0000   (1),

wherein X₁ represents a peak area value of the first peptide fragmentcalculated by an automatic integration method from an extracted ionchromatogram obtained by an LC-MS/MS analysis of the composition, and Yis the same as defined above.

-   [10] The composition according to [8] or [9], wherein:

the peptide fragment group (B) contains the second peptide fragment; and

a content ratio of the second peptide fragment to the alkalinephosphatase satisfies formula (2):

(X ₂ /Y)×100≤1.6000   (2),

wherein X₂ represents a peak area value of the second peptide fragmentcalculated by an automatic integration method from an extracted ionchromatogram obtained by an LC-MS/MS analysis of the composition, and Yis the same as defined above.

-   [11] A composition containing:

an alkaline phosphatase; and

a peptide fragment group (C) composed of two or more peptide fragments,wherein each of the two or more peptide fragments consists of 12 to 50consecutive amino acid residues selected from positions 501 to 578 ofthe amino acid sequence set forth in SEQ ID NO: 5 and contains positions534 to 545 of the amino acid sequence set forth in SEQ ID NO: 5,

wherein a content ratio of the peptide fragment group (C) to thealkaline phosphatase satisfies formula (C):

(X _(C) /Y)×100≤1.0000   (C),

wherein X_(C) represents a peak area value of the peptide fragment group(C) calculated by an automatic integration method from an extracted ionchromatogram obtained by an LC-MS/MS analysis of the composition, and Yrepresents a peak area value of the alkaline phosphatase calculated byan automatic integration method from a chromatogram obtained by an LC-UVanalysis of the composition.

-   [12] The composition according to [11], wherein the peptide fragment    group (C) contains one or two peptide fragments selected from the    group consisting of a third peptide fragment consisting of the amino    acid sequence set forth in SEQ ID NO: 3 and a fourth peptide    fragment consisting of the amino acid sequence set forth in SEQ ID    NO: 4.-   [13] The composition according to [12], wherein:

the peptide fragment group (C) contains the third peptide fragment; and

a content ratio of the third peptide fragment to the alkalinephosphatase satisfies formula (3):

(X ₃ /Y)×100≤0.2000   (3),

wherein X₃ represents a peak area value of the third peptide fragmentcalculated by an automatic integration method from an extracted ionchromatogram obtained by an LC-MS/MS analysis of the composition, and Yis the same as defined above.

-   [14] The composition according to [12] or [13], wherein:

the peptide fragment group (C) contains the fourth peptide fragment; and

a content ratio of the fourth peptide fragment to the alkalinephosphatase satisfies formula (4):

(X ₄ /Y)×100≤0.3500   (4),

wherein X₄ represents a peak area value of the fourth peptide fragmentcalculated by an automatic integration method from an extracted ionchromatogram obtained by an LC-MS/MS analysis of the composition, and Yis the same as defined above.

-   [15] A composition containing:

an alkaline phosphatase; and

a second peptide fragment consisting of the amino acid sequence setforth in SEQ ID NO: 2,

wherein a content ratio of the second peptide fragment to the alkalinephosphatase satisfies formula (2):

(X ₂ /Y)×100≤1.6000   (2),

wherein X₂ represents a peak area value of the second peptide fragmentcalculated by an automatic integration method from an extracted ionchromatogram obtained by an LC-MS/MS analysis of the composition, and Yrepresents a peak area value of the alkaline phosphatase calculated byan automatic integration method from a chromatogram obtained by an LC-UVanalysis of the composition.

-   [16] The composition according to [15], wherein:

the composition further contains a first peptide fragment consisting ofthe amino acid sequence set forth in SEQ ID NO: 1; and

a content ratio of the first peptide fragment to the alkalinephosphatase satisfies formula (1):

(X ₁ /Y)×100≤1.0000   (1),

wherein X₁ represents a peak area value of the first peptide fragmentcalculated by an automatic integration method from an extracted ionchromatogram obtained by an LC-MS/MS analysis of the composition, and Yis the same as defined above.

-   [17] The composition according to [15] or [16], wherein:

the composition further contains a third peptide fragment consisting ofthe amino acid sequence set forth in SEQ ID NO: 3; and

a content ratio of the third peptide fragment to the alkalinephosphatase satisfies formula (3):

(X ₃ /Y)×100≤0.2000   (3),

wherein X₃ represents a peak area value of the third peptide fragmentcalculated by an automatic integration method from an extracted ionchromatogram obtained by an LC-MS/MS analysis of the composition, and Yis the same as defined above.

-   [18] The composition according to any one of [15] to [17], wherein:

the composition further contains a fourth peptide fragment consisting ofthe amino acid sequence set forth in SEQ ID NO: 4; and

a content ratio of the fourth peptide fragment to the alkalinephosphatase satisfies formula (4):

(X ₄ /Y)×100≤0.3500   (4),

wherein X₄ represents a peak area value of the fourth peptide fragmentcalculated by an automatic integration method from an extracted ionchromatogram obtained by an LC-MS/MS analysis of the composition, and Yis the same as defined above.

-   [19] A composition containing:

an alkaline phosphatase; and

a third peptide fragment consisting of the amino acid sequence set forthin SEQ ID NO: 3,

wherein a content ratio of the third peptide fragment to the alkalinephosphatase satisfies formula (3):

(X ₃ /Y)×100≤0.2000   (3),

wherein X₃ represents a peak area value of the third peptide fragmentcalculated by an automatic integration method from an extracted ionchromatogram obtained by an LC-MS/MS analysis of the composition, and Yis the same as defined above.

-   [20] The composition according to [19], wherein:

the composition further contains a first peptide fragment consisting ofthe amino acid sequence set forth in SEQ ID NO: 1; and

a content ratio of the first peptide fragment to the alkalinephosphatase satisfies formula (1):

(X ₁ /Y)×100≤1.0000   (1),

wherein X₁ represents a peak area value of the first peptide fragmentcalculated by an automatic integration method from an extracted ionchromatogram obtained by an LC-MS/MS analysis of the composition, and Yis the same as defined above.

-   [21] The composition according to [19] or [20], wherein:

the composition further contains a fourth peptide fragment consisting ofthe amino acid sequence set forth in SEQ ID NO: 4; and

a content ratio of the fourth peptide fragment to the alkalinephosphatase satisfies formula (4):

(X ₄ /Y)×100≤0.3500   (4),

wherein X₄ represents a peak area value of the fourth peptide fragmentcalculated by an automatic integration method from an extracted ionchromatogram obtained by an LC-MS/MS analysis of the composition, and Yis the same as defined above.

-   [22] A composition containing:

an alkaline phosphatase; and

a fourth peptide fragment consisting of the amino acid sequence setforth in SEQ ID NO: 4,

wherein a content ratio of the fourth peptide fragment to the alkalinephosphatase satisfies formula (4):

(X ₄ /Y)×100≤0.3500   (4),

wherein X₄ represents a peak area value of the fourth peptide fragmentcalculated by an automatic integration method from an extracted ionchromatogram obtained by an LC-MS/MS analysis of the composition, and Yis the same as defined above.

-   [23] The composition according to [22], wherein:

the composition further contains a first peptide fragment consisting ofthe amino acid sequence set forth in SEQ ID NO: 1; and

a content ratio of the first peptide fragment to the alkalinephosphatase satisfies formula (1):

(X ₁ /Y)×100≤1.0000   (1),

wherein X₁ represents a peak area value of the first peptide fragmentcalculated by an automatic integration method from an extracted ionchromatogram obtained by an LC-MS/MS analysis of the composition, and Yis the same as defined above.

-   [24] The composition according to any one of [1] to [23], wherein    the composition has an alkaline phosphatase specific activity of    2,000 U/mg or more.-   [25] The composition according to any one of [1] to [24], wherein    the alkaline phosphatase is selected from the following (a) and (b):-   (a) an alkaline phosphatase containing a protein molecule consisting    of the amino acid sequence set forth in SEQ ID NO: 5; and-   (b) an alkaline phosphatase containing a protein molecule consisting    of an amino acid sequence that has 70% or more sequence identity to    the amino acid sequence set forth in SEQ ID NO: 5 and comprises    positions 501 to 578 of the amino acid sequence set forth in SEQ ID    NO: 5.-   [26] The composition according to any one of [1] to [25], wherein    the composition further contains a nucleic acid.-   [27] The composition according to [26], wherein the composition is a    composition used for dephosphorylating the nucleic acid.-   [28] The composition according to any one of [1] to [25], wherein    the composition further contains a dephosphorylated nucleic acid.-   [29] The composition according to [28], wherein the composition is a    composition used for preparing a labeled nucleic acid containing the    dephosphorylated nucleic acid and a labeling substance bound to the    dephosphorylated nucleic acid.-   [30] The composition according to any one of [1] to [25], wherein    the composition further contains a labeled nucleic acid containing a    dephosphorylated nucleic acid and a labeling substance bound to the    dephosphorylated nucleic acid.-   [31] The composition according to [30], wherein the composition is a    nucleic acid sample to be subjected to a nucleic acid detection    method.-   [32] The composition according to [31], wherein the nucleic acid    detection method is a nucleic acid detection method using a nucleic    acid microarray.-   [33] A method of producing a dephosphorylated nucleic acid, the    method including the following steps of:

providing the composition according to any one of [1] to [25];

providing a nucleic acid; and

treating the nucleic acid with the composition to dephosphorylate thenucleic acid.

-   [34] A method of producing a labeled nucleic acid, the method    including the following steps of:

providing the composition according to any one of [1] to [25];

providing a nucleic acid;

providing a labeling substance;

treating the nucleic acid with the composition to dephosphorylate thenucleic acid; and

binding the labeling substance to the dephosphorylated nucleic acid.

We thus provide a composition containing an alkaline phosphatase andhaving a high quality, a method of producing a dephosphorylated nucleicacid by using the composition and a method of producing a labelednucleic acid by using the composition.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an extracted ion chromatogram on the first peptide fragmentobtained by an LC-MS/MS analysis of the composition C2 in ComparativeExample 2.

FIG. 2 shows an extracted ion chromatogram on the second peptidefragment obtained by an LC-MS/MS analysis of the composition C2 inComparative Example 2.

FIG. 3 shows an extracted ion chromatogram on the third peptide fragmentobtained by an LC-MS/MS analysis of the composition C2 in ComparativeExample 2.

FIG. 4 shows an extracted ion chromatogram on the fourth peptidefragment obtained by an LC-MS/MS analysis of the composition C2 inComparative Example 2.

FIG. 5 shows an extracted ion chromatogram on the first peptide fragmentobtained by an LC-MS/MS analysis of the composition E1 (purified productof the composition C2) in Example 1.

FIG. 6 shows an extracted ion chromatogram on the second peptidefragment obtained by an LC-MS/MS analysis of the composition E1(purified product of the composition C2) in Example 1.

FIG. 7 shows an extracted ion chromatogram on the third peptide fragmentobtained by an LC-MS/MS analysis of the composition E1 (purified productof the composition C2) in Example 1.

FIG. 8 shows an extracted ion chromatogram on the fourth peptidefragment obtained by an LC-MS/MS analysis of the composition E1(purified product of the composition C2) in Example 1.

FIG. 9 shows a chromatogram on an alkaline phosphatase obtained by anLC-UV analysis of the composition E1 (purified product of thecomposition C2) in Example 1.

DETAILED DESCRIPTION

Our compositions and methods will be described in detail below. It ispossible to combine two or more of the aspects described below, and itis possible to combine two or more of the examples described below. Thisdisclosure also encompasses such combinations. The expression “numericalvalue M to numerical value N” means a range of numerical value M or moreand numerical value N or less.

We provide a composition containing an alkaline phosphatase and apeptide fragment group (A) as a first composition.

We provide a composition containing an alkaline phosphatase and apeptide fragment group (B) as a second composition.

We provide a composition containing an alkaline phosphatase and apeptide fragment group (C) as a third composition.

We provide a composition containing an alkaline phosphatase and a secondpeptide fragment consisting of the amino acid sequence set forth in SEQID NO: 2 as a fourth composition.

We provide a composition containing an alkaline phosphatase and a thirdpeptide fragment consisting of the amino acid sequence set forth in SEQID NO: 3 as a fifth composition.

We provide a composition containing an alkaline phosphatase and a fourthpeptide fragment consisting of the amino acid sequence set forth in SEQID NO: 4 as a sixth composition.

The compositions above will be collectively expressed as the“composition.” Therefore, the descriptions of the “composition” apply toany of the above compositions unless otherwise specified.

Alkaline Phosphatase

The composition contains an alkaline phosphatase. The composition maycontain one alkaline phosphatase or may contain two or more alkalinephosphatases.

The alkaline phosphatase contained in the composition is notparticularly limited as long as it has alkaline phosphatase activity.The alkaline phosphatase activity is activity that hydrolyzes aphosphoric monoester bond in alkalinity (pH 8 to 11, e.g., pH 8 to 10 orpH 9 to 11), and the reaction form is classified into EC3.1.3.1.

The structure of the alkaline phosphatase contained in the composition(e.g., primary structure, secondary structure, tertiary structure,quaternary structure and the like) is not particularly limited. Forexample, the alkaline phosphatase may have a sugar chain or may not havea sugar chain. The alkaline phosphatase may be any isozyme that canexist based on differences in the structure of a protein molecule (e.g.,amino acid sequence of a protein molecule), glycosylation and the like.The alkaline phosphatase may be a monomer that is formed from onesubunit or may be an oligomer that is formed from two or more subunits(e.g., dimer, tetramer and the like). The oligomer may be a homooligomeror may be a heterooligomer.

The animal from which the alkaline phosphatase contained in thecomposition is derived is not particularly limited. Examples of theanimal from which the alkaline phosphatase is derived include a bovine,a shrimp, a microorganism into which a gene encoding an alkalinephosphatase has been introduced and the like. Since a bovine-derivedalkaline phosphatase has high alkaline phosphatase activity, the animalfrom which the alkaline phosphatase is derived is preferably a bovine.When the alkaline phosphatase is derived from a bovine, the organ fromwhich the alkaline phosphatase is derived is preferably small intestineor large intestine.

The alkaline phosphatase contained in the composition may be wild-typeor may be mutated. The mutated alkaline phosphatase contains, forexample, a protein molecule consisting of an amino acid sequenceobtained by introducing deletion, substitution, insertion or addition ofone or more amino acids to an amino acid sequence of a protein moleculeof a wild-type alkaline phosphatase. The amino acid sequence of theprotein molecule of the mutated alkaline phosphatase has preferably 70%or more, more preferably 75% or more, still more preferably 80% or more,yet more preferably 85% or more, further preferably 90% or more, andstill further preferably 95% or more sequence identity to the amino acidsequence of the protein molecule of the wild-type alkaline phosphatase.

Preferably, the alkaline phosphatase is selected from (a) and (b):

-   (a) an alkaline phosphatase containing a protein molecule consisting    of the amino acid sequence set forth in SEQ ID NO: 5; and-   (b) an alkaline phosphatase containing a protein molecule consisting    of an amino acid sequence that has 70% or more sequence identity to    the amino acid sequence set forth in SEQ ID NO: 5 and contains    positions 501 to 578 of the amino acid sequence set forth in SEQ ID    NO: 5. In this example, the composition may contain one alkaline    phosphatase selected from (a) and (b), or may contain two or more    alkaline phosphatases selected from (a) and (b).

The amino acid sequence of the protein molecule of the alkalinephosphatase (a) (i.e., the amino acid sequence set forth in SEQ ID NO:5) corresponds to an amino acid sequence of a protein molecule of abovine-derived alkaline phosphatase. Therefore, a bovine-derivedalkaline phosphatase falls within the alkaline phosphatase (a).

The amino acid sequence of the protein molecule of the alkalinephosphatase (b) has preferably 70% or more, more preferably 75% or more,still more preferably 80% or more, yet more preferably 85% or more,further preferably 90% or more, and still further preferably 95% or moresequence identity to the amino acid sequence set forth in SEQ ID NO: 5.

Both of a wild-type alkaline phosphatase (e.g., an alkaline phosphatasederived from an animal other than a bovine, a bovine-derived alkalinephosphatase having a polymorphism or the like) and a mutated alkalinephosphatase fall within the alkaline phosphatase (b). The position(s) atwhich one or more amino acids are deleted, substituted, inserted oradded in the amino acid sequence set forth in SEQ ID NO: 5 is/are aposition(s) other than positions 501 to 578 of the amino acid sequenceset forth in SEQ ID NO: 5.

The alkaline phosphatases (a) and (b) can generate the peptide fragmentgroup (A), the peptide fragment group (B), the peptide fragment group(C), the second peptide fragment, the third peptide fragment, the fourthpeptide fragment and the like, by decomposition of the alkalinephosphatases.

Peptide First Group (A)

The first composition may contain the peptide fragment group (A). Thepeptide fragment group (A) is composed of two or more peptide fragments,and each peptide fragment constituting the peptide fragment group (A)consists of 5 to 50 consecutive amino acid residues selected frompositions 501 to 578 of the amino acid sequence set forth in SEQ ID NO:5. The peptide fragment group (A) is composed of, among peptidefragments contained in the first composition, all peptide fragments eachcorresponding to a peptide fragment consisting of 5 to 50 consecutiveamino acid residues selected from positions 501 to 578 of the amino acidsequence set forth in SEQ ID NO: 5. Preferably, the peptide fragmentgroup (A) is composed of three or more peptide fragments. Furtherpreferably, the peptide fragment group (A) is composed of four or morepeptide fragments.

The amino acid sequence of each peptide fragment constituting thepeptide fragment group (A) is a part (moiety consisting of a pluralityof consecutive amino acid residues) of positions 501 to 578 of the aminoacid sequence set forth in SEQ ID NO: 5. In other words, the peptidefragment group (A) can be generated by decomposition of positions 501 to578 of the amino acid sequence set forth in SEQ ID NO: 5. This does notmean that the alkaline phosphatase contained in the first composition isrequired to contain positions 501 to 578 of the amino acid sequence setforth in SEQ ID NO: 5. The alkaline phosphatase contained in the firstcomposition may not contain positions 501 to 578 of the amino acidsequence set forth in SEQ ID NO: 5. However, the alkaline phosphatasecontained in the first composition preferably contains positions 501 to578 of the amino acid sequence set forth in SEQ ID NO: 5.

The amino acid sequence of each peptide fragment constituting thepeptide fragment group (A) is not particularly limited as long as theamino acid sequence is a part of positions 501 to 578 of the amino acidsequence set forth in SEQ ID NO: 5. The amino acid sequence of eachpeptide fragment constituting the peptide fragment group (A) ispreferably a part of positions 505 to 578 of the amino acid sequence setforth in SEQ ID NO: 5, more preferably a part of positions 511 to 578 ofthe amino acid sequence set forth in SEQ ID NO: 5, and still morepreferably a part of positions 511 to 571 of the amino acid sequence setforth in SEQ ID NO: 5.

The number of amino acid residues constituting each peptide fragmentconstituting the peptide fragment group (A) is not particularly limitedas long as the number is 5 to 50, and the number is preferably 5 to 45,more preferably 10 to 40, and still more preferably 10 to 30.

Preferably, the peptide fragment group (A) contains one, two, three orfour peptide fragments selected from the group consisting of the firstpeptide fragment consisting of the amino acid sequence set forth in SEQID NO: 1, the second peptide fragment consisting of the amino acidsequence set forth in SEQ ID NO: 2, the third peptide fragmentconsisting of the amino acid sequence set forth in SEQ ID NO: 3 and thefourth peptide fragment consisting of the amino acid sequence set forthin SEQ ID NO: 4. In this example, the peptide fragment group (A) may ormay not contain a peptide fragment other than the first to fourthpeptide fragments.

The amino acid sequence set forth in SEQ ID NO: 1 (VPLASETHGGEDVAVF)corresponds to positions 516 to 531 of the amino acid sequence set forthin SEQ ID NO: 5. The amino acid sequence set forth in SEQ ID NO: 2(VPLASETHGGEDV) corresponds to positions 516 to 528 of the amino acidsequence set forth in SEQ ID NO: 5. The amino acid sequence set forth inSEQ ID NO: 3 (GPQAHLVHGVQEETFVAH) corresponds to positions 534 to 551 ofthe amino acid sequence set forth in SEQ ID NO: 5. The amino acidsequence set forth in SEQ ID NO: 4 (GPQAHLVHGVQE) corresponds topositions 534 to 545 of the amino acid sequence set forth in SEQ ID NO:5.

The peptide fragment group (A) can be generated by decomposition of analkaline phosphatase containing positions 501 to 578 of the amino acidsequence set forth in SEQ ID NO: 5. The peptide fragment group (A) maybe one generated by decomposition of an alkaline phosphatase notcontained in the first composition, but is usually one generated bydecomposition of an alkaline phosphatase contained in the firstcomposition. Therefore, preferably, the alkaline phosphatase containedin the first composition is an alkaline phosphatase that can generatethe peptide fragment group (A). Preferably, the alkaline phosphatasethat can generate the peptide fragment group (A) is selected from thealkaline phosphatases (a) and (b). In this example, the firstcomposition contains one or two or more alkaline phosphatases selectedfrom the alkaline phosphatases (a) and (b).

Peptide Second Group (B)

The second composition contains the peptide fragment group (B). Thepeptide fragment group (B) is composed of two or more peptide fragments,and each peptide fragment constituting the peptide fragment group (B)consists of 13 to 50 consecutive amino acid residues selected frompositions 501 to 578 of the amino acid sequence set forth in SEQ ID NO:5 and contains positions 516 to 528 (VPLASETHGGEDV) of the amino acidsequence set forth in SEQ ID NO: 5. The peptide fragment group (B) iscomposed of, among peptide fragments contained in the secondcomposition, all peptide fragments each corresponding to a peptidefragment consisting of 13 to 50 consecutive amino acid residues selectedfrom positions 501 to 578 of the amino acid sequence set forth in SEQ IDNO: 5 and containing positions 516 to 528 of the amino acid sequence setforth in SEQ ID NO: 5.

In the amino acid sequence of each peptide fragment constituting thepeptide fragment group (B), the position at which positions 516 to 528of the amino acid sequence set forth in SEQ ID NO: 5 are contained isnot particularly limited. The position at which positions 516 to 528 ofthe amino acid sequence set forth in SEQ ID NO: 5 are contained may beany of the N terminal part of a peptide fragment, the C terminal part ofa peptide fragment, and a part other than the N terminal part and the Cterminal part of a peptide fragment.

The amino acid sequence of each peptide fragment constituting thepeptide fragment group (B) is a part (moiety consisting of a pluralityof consecutive amino acid residues) of positions 501 to 578 of the aminoacid sequence set forth in SEQ ID NO: 5. In other words, the peptidefragment group (B) can be generated by decomposition of positions 501 to578 of the amino acid sequence set forth in SEQ ID NO: 5. This does notmean that the alkaline phosphatase contained in the second compositionis required to contain positions 501 to 578 of the amino acid sequenceset forth in SEQ ID NO: 5. The alkaline phosphatase contained in thesecond composition may not contain positions 501 to 578 of the aminoacid sequence set forth in SEQ ID NO: 5. However, the alkalinephosphatase contained in the second composition preferably containspositions 501 to 578 of the amino acid sequence set forth in SEQ ID NO:5.

The amino acid sequence of each peptide fragment constituting thepeptide fragment group (B) is not particularly limited as long as theamino acid sequence is a part of positions 501 to 578 of the amino acidsequence set forth in SEQ ID NO: 5 and contains positions 516 to 528 ofthe amino acid sequence set forth in SEQ ID NO: 5. The amino acidsequence of each peptide fragment constituting the peptide fragmentgroup (B) is preferably a part of positions 501 to 570 of the amino acidsequence set forth in SEQ ID NO: 5, more preferably a part of positions501 to 560 of the amino acid sequence set forth in SEQ ID NO: 5, andstill more preferably a part of positions 501 to 555 of the amino acidsequence set forth in SEQ ID NO: 5.

The number of amino acid residues constituting each peptide fragmentconstituting the peptide fragment group (B) is not particularly limitedas long as the number is 13 to 50, and the number is preferably 13 to45, more preferably 13 to 40, and still more preferably 13 to 35.

Preferably, the peptide fragment group (B) contains one or two peptidefragments selected from the group consisting of the first peptidefragment consisting of the amino acid sequence set forth in SEQ ID NO: 1and the second peptide fragment consisting of the amino acid sequenceset forth in SEQ ID NO: 2. In this example, the peptide fragment group(B) may or may not contain a peptide fragment other than the first andsecond peptide fragments.

The amino acid sequence set forth in SEQ ID NO: 1 (VPLASETHGGEDVAVF)corresponds to positions 516 to 531 of the amino acid sequence set forthin SEQ ID NO: 5 and contains positions 516 to 528 of the amino acidsequence set forth in SEQ ID NO: 5 (the underlined part corresponds topositions 516 to 528 of the amino acid sequence set forth in SEQ ID NO:5). The amino acid sequence set forth in SEQ ID NO: 2 (VPLASETHGGEDV)corresponds to positions 516 to 528 of the amino acid sequence set forthin SEQ ID NO: 5.

The peptide fragment group (B) can be generated by decomposition of analkaline phosphatase containing positions 501 to 578 of the amino acidsequence set forth in SEQ ID NO: 5. The peptide fragment group (B) maybe one generated by decomposition of an alkaline phosphatase notcontained in the second composition, but is usually one generated bydecomposition of an alkaline phosphatase contained in the secondcomposition. Therefore, preferably, the alkaline phosphatase containedin the second composition is an alkaline phosphatase that can generatethe peptide fragment group (B). Preferably, the alkaline phosphatasethat can generate the peptide fragment group (B) is selected from thealkaline phosphatases (a) and (b). In this example, the secondcomposition contains one or two or more alkaline phosphatases selectedfrom the alkaline phosphatases (a) and (b).

Peptide Third Group (C)

The third composition contains a peptide fragment group (C). The peptidefragment group (C) is composed of two or more peptide fragments, andeach peptide fragment constituting the peptide fragment group (C)consists of 12 to 50 consecutive amino acid residues selected frompositions 501 to 578 of the amino acid sequence set forth in SEQ ID NO:5 and contains positions 534 to 545 (GPQAHLVHGVQE) of the amino acidsequence set forth in SEQ ID NO: 5. The peptide fragment group (C) iscomposed of, among peptide fragments contained in the third composition,all peptide fragments each corresponding to a peptide fragmentconsisting of 12 to 50 consecutive amino acid residues selected frompositions 501 to 578 of the amino acid sequence set forth in SEQ ID NO:5 and containing positions 534 to 545 of the amino acid sequence setforth in SEQ ID NO: 5.

In the amino acid sequence of each peptide fragment constituting thepeptide fragment group (C), the position at which positions 534 to 545of the amino acid sequence set forth in SEQ ID NO: 5 may be any of the Nterminal part of a peptide fragment, the C terminal part of a peptidefragment, and a part other than the N terminal part and the C terminalpart of a peptide fragment.

The amino acid sequence of each peptide fragment constituting thepeptide fragment group (C) is a part (moiety consisting of a pluralityof consecutive amino acid residues) of positions 501 to 578 of the aminoacid sequence set forth in SEQ ID NO: 5. In other words, the peptidefragment group (C) can be generated by decomposition of positions 501 to578 of the amino acid sequence set forth in SEQ ID NO: 5. This does notmean that the alkaline phosphatase contained in the third composition isrequired to contain positions 501 to 578 of the amino acid sequence setforth in SEQ ID NO: 5. The alkaline phosphatase contained in the thirdcomposition may not contain positions 501 to 578 of the amino acidsequence set forth in SEQ ID NO: 5. However, the alkaline phosphatasecontained in the third composition preferably contains positions 501 to578 of the amino acid sequence set forth in SEQ ID NO: 5.

The amino acid sequence of each peptide fragment constituting thepeptide fragment group (C) is not particularly limited as long as theamino acid sequence is a part of positions 501 to 578 of the amino acidsequence set forth in SEQ ID NO: 5 and contains positions 534 to 545 ofthe amino acid sequence set forth in SEQ ID NO: 5. The amino acidsequence of each peptide fragment constituting the peptide fragmentgroup (C) is preferably a part of positions 506 to 578 of the amino acidsequence set forth in SEQ ID NO: 5, more preferably a part of positions511 to 578 of the amino acid sequence set forth in SEQ ID NO: 5, andstill more preferably a part of positions 521 to 578 of the amino acidsequence set forth in SEQ ID NO: 5.

The number of amino acid residues constituting each peptide fragmentconstituting the peptide fragment group (C) is not particularly limitedas long as the number is 12 to 50, and the number is preferably 12 to45, more preferably 12 to 40, and still more preferably 12 to 35.

Preferably, the peptide fragment group (C) contains one or two peptidefragments selected from the group consisting of the third peptidefragment consisting of the amino acid sequence set forth in SEQ ID NO: 3and the fourth peptide fragment consisting of the amino acid sequenceset forth in SEQ ID NO: 4. In this example, the peptide fragment group(C) may or may not contain a peptide fragment other than the third andfourth peptide fragments.

The amino acid sequence set forth in SEQ ID NO: 3 (GPQAHLVHGVQEETFVAH)corresponds to positions 534 to 551 of the amino acid sequence set forthin SEQ ID NO: 5 and contains positions 534 to 545 of the amino acidsequence set forth in SEQ ID NO: 5 (the underlined part corresponds topositions 534 to 545 of the amino acid sequence set forth in SEQ ID NO:5). The amino acid sequence set forth in SEQ ID NO: 4 (GPQAHLVHGVQE)corresponds to positions 534 to 545 of the amino acid sequence set forthin SEQ ID NO: 5.

The peptide fragment group (C) can be generated by decomposition of analkaline phosphatase containing positions 501 to 578 of the amino acidsequence set forth in SEQ ID NO: 5. The peptide fragment group (C) maybe one generated by decomposition of an alkaline phosphatase notcontained in the third composition, but is usually one generated bydecomposition of an alkaline phosphatase contained in the thirdcomposition. Therefore, preferably, the alkaline phosphatase containedin the third composition is an alkaline phosphatase that can generatethe peptide fragment group (C). Preferably, the alkaline phosphatasethat can generate the peptide fragment group (C) is selected from thealkaline phosphatases (a) and (b). In this example, the thirdcomposition contains one or two or more alkaline phosphatases selectedfrom the alkaline phosphatases (a) and (b).

First Peptide Fragment

Preferably, the first, second, third, fourth, fifth or sixthcompositions contain the first peptide fragment consisting of the aminoacid sequence set forth in SEQ ID NO: 1.

The first peptide fragment can be generated by decomposition of analkaline phosphatase containing positions 501 to 578 of the amino acidsequence set forth in SEQ ID NO: 5. The first peptide fragment may beone generated by decomposition of an alkaline phosphatase not containedin the first, second, third, fourth, fifth or sixth compositions, but isusually one generated by decomposition of an alkaline phosphatasecontained in the first, second, third, fourth, fifth or sixthcompositions. Therefore, preferably, the alkaline phosphatase containedin the first, second, third, fourth, fifth or sixth compositions is analkaline phosphatase that can generate the first peptide fragment.Preferably, the alkaline phosphatase that can generate the first peptidefragment is selected from the alkaline phosphatases (a) and (b). In thisexample, the first, second, third, fourth, fifth or sixth compositionscontain one or two or more alkaline phosphatases selected from thealkaline phosphatases (a) and (b).

Second Peptide Fragment

The fourth composition contains the second peptide fragment consistingof the amino acid sequence set forth in SEQ ID NO: 2. Preferably, thefourth composition further contains one, two or three peptide fragmentsselected from the group consisting of the first peptide fragmentconsisting of the amino acid sequence set forth in SEQ ID NO: 1, thethird peptide fragment consisting of the amino acid sequence set forthin SEQ ID NO: 3 and the fourth peptide fragment consisting of the aminoacid sequence set forth in SEQ ID NO: 4. In this example, the fourthcomposition may or may not contain a peptide fragment other than thefirst to fourth peptide fragments.

The second peptide fragment can be generated by decomposition of analkaline phosphatase containing positions 501 to 578 of the amino acidsequence set forth in SEQ ID NO: 5. The second peptide fragment may beone generated by decomposition of an alkaline phosphatase not containedin the fourth composition, but is usually one generated by decompositionof an alkaline phosphatase contained in the fourth composition.Therefore, preferably, the alkaline phosphatase contained in the fourthcomposition is an alkaline phosphatase that can generate the secondpeptide fragment. Preferably, the alkaline phosphatase that can generatethe second peptide fragment is selected from the alkaline phosphatases(a) and (b). In this example, the fourth composition contains one or twoor more alkaline phosphatases selected from the alkaline phosphatases(a) and (b).

Third Peptide Fragment

The fifth composition contains the third peptide fragment consisting ofthe amino acid sequence set forth in SEQ ID NO: 3. Preferably, the fifthcomposition further contains one, two or three peptide fragmentsselected from the group consisting of the first peptide fragmentconsisting of the amino acid sequence set forth in SEQ ID NO: 1, thesecond peptide fragment consisting of the amino acid sequence set forthin SEQ ID NO: 2 and the fourth peptide fragment consisting of the aminoacid sequence set forth in SEQ ID NO: 4. In this example, the fifthcomposition may or may not contain a peptide fragment other than thefirst to fourth peptide fragments.

The third peptide fragment can be generated by decomposition of analkaline phosphatase containing positions 501 to 578 of the amino acidsequence set forth in SEQ ID NO: 5. The third peptide fragment may beone generated by decomposition of an alkaline phosphatase not containedin the fifth composition, but is usually one generated by decompositionof an alkaline phosphatase contained in the fifth composition.Therefore, preferably, the alkaline phosphatase contained in the fifthcomposition is an alkaline phosphatase that can generate the thirdpeptide fragment. Preferably, the alkaline phosphatase that can generatethe third peptide fragment is selected from the alkaline phosphatases(a) and (b). In this example, the fifth composition contains one or twoor more alkaline phosphatases selected from the alkaline phosphatases(a) and (b).

Fourth Peptide Fragment

The sixth composition contains the fourth peptide fragment consisting ofthe amino acid sequence set forth in SEQ ID NO: 4. Preferably, the sixthcomposition further contains one, two or three peptide fragmentsselected from the group consisting of the first peptide fragmentconsisting of the amino acid sequence set forth in SEQ ID NO: 1, thesecond peptide fragment consisting of the amino acid sequence set forthin SEQ ID NO: 2 and the third peptide fragment consisting of the aminoacid sequence set forth in SEQ ID NO: 3. In this example, the sixthcomposition may or may not contain a peptide fragment other than thefirst to fourth peptide fragments.

The fourth peptide fragment can be generated by decomposition of analkaline phosphatase containing positions 501 to 578 of the amino acidsequence set forth in SEQ ID NO: 5. The fourth peptide fragment may beone generated by decomposition of an alkaline phosphatase not containedin the sixth composition, but is usually one generated by decompositionof an alkaline phosphatase contained in the sixth composition.Therefore, preferably, the alkaline phosphatase contained in the sixthcomposition is an alkaline phosphatase that can generate the fourthpeptide fragment. Preferably, the alkaline phosphatase that can generatethe fourth peptide fragment is selected from the alkaline phosphatases(a) and (b). In this example, the sixth composition contains one or twoor more alkaline phosphatases selected from the alkaline phosphatases(a) and (b).

Content Ratio of Peptide Fragment Group (A) to Alkaline Phosphatase

In the first composition, the content ratio of the peptide fragmentgroup (A) to the alkaline phosphatase satisfies formula (A):

(X _(A) /Y)×100≤4.4000   (A).

In formula (A), X_(A) represents a peak area value of the peptidefragment group (A) calculated by an automatic integration method from anextracted ion chromatogram obtained by an LC-MS/MS analysis of the firstcomposition, and Y represents a peak area value of the alkalinephosphatase calculated by an automatic integration method from achromatogram obtained by an LC-UV analysis of the first composition. The“peak area value of the peptide fragment group (A)” means a total peakarea value of all peptide fragments constituting the peptide fragmentgroup (A). The “peak area value of the alkaline phosphatase” means, whenthe first composition contains one alkaline phosphatase, a peak areavalue of the one alkaline phosphatase, and, when the first compositioncontains two or more alkaline phosphatases, a total peak area value ofthe two or more alkaline phosphatases (i.e., total peak area value ofall alkaline phosphatases contained in the first composition).

The LC-MS/MS analysis and the LC-UV analysis are performed by using asample in which the content ratio of the peptide fragment group (A) tothe alkaline phosphatase is the same as that of the first composition.The LC-MS/MS analysis and the LC-UV analysis can be performed, forexample, by using an aqueous solution prepared from the firstcomposition and with an alkaline phosphatase concentration of 10% byweight.

The LC-MS/MS analysis is one of the hyphenated methods. The hyphenatedmethod is a method of analyzing by connecting a chromatograph such as agas chromatograph and a liquid chromatograph to a mass spectrometer. TheLC-MS/MS analysis is a method of analyzing by connecting a liquidchromatograph (LC) to a tandem mass spectrometer (MS/MS). In theLC-MS/MS analysis, analyte components separated by the liquidchromatograph are ionized, the ions thus produced are separated by thetandem mass spectrometer, and specific mass ions are fragmented anddetected.

In the hyphenated method, an extracted ion chromatogram is achromatogram expressed as a function of time obtained by measuring amass spectrum at a certain time interval and storing it in a computer,followed by reading a relative intensity at a specific (not necessarilyone type) m/z value. The m/z value of an ion of each peptide fragmentconstituting the peptide fragment group (A) used for detecting a peak ofeach peptide fragment constituting the peptide fragment group (A) ispreferably 50 to 2,200, more preferably 200 to 1,500, and still morepreferably 300 to 1,200. Regarding an ion of a peptide fragment with anm/z value being specified, it is possible to create an extracted ionchromatogram of the ion of the peptide fragment based on the m/z value.The m/z value of the first peptide fragment is 814.4018, the m/z valueof the second peptide fragment is 655.8148, the m/z value of the thirdpeptide fragment is 652.6623, and the m/z value of the fourth peptidefragment is 636.3282. Regarding a peptide fragment with an m/z value notbeing specified, after whether a certain peak corresponds to a peak of apredetermined peptide fragment or not is confirmed by an amino acidsequence analysis of a peptide fragment showing the peak, it is possibleto create an extracted ion chromatogram of the peptide fragment based onthe m/z value of the peak.

The LC-UV analysis is a method of analyzing by connecting a liquidchromatograph (LC) to an ultraviolet detector (UV detector). In theLC-UV analysis, an alkaline phosphatase is detected as a componenthaving absorption at 214 nm.

Conditions of the LC-MS/MS analysis are as follows.

Conditions of LC-MS/MS Analysis Apparatus Configuration

Mass spectrometer: maXis impact (manufactured by Bruker Daltnics, Inc.)

Conditions of Mass Spectrometry

Ionization method: ESI

Measured ion: cation

Capillary voltage: 4,500 V

Nebulizer: 2.0 bar

Dry gas: 8.0 L/min

Detector voltage: 1,823 V

Measuring span (MS): m/z 50 to 2,200

MS/MS Conditions

Measuring span (MS): m/z 50 to 2,200

Collision gas: nitrogen

Conditions of LC-UV Analysis Apparatus Configuration

Liquid chromatograph: LC-30A system (manufactured by ShimadzuCorporation)

Detector: UV-Vis (190 to 900 nm, manufactured by Shimadzu Corporation)

Conditions of Liquid Chromatography

Column: Acquity BEH C18 1.7 μm (manufactured by Waters Corporation)

Column size: 2.1 mm×100 mm

Column temperature: 50° C.

Mobile phase flow rate: 0.2 mL/min

Mobile phase A: mixed solution of water/formic acid (1000:1)

Mobile phase B: mixed solution of acetonitrile/water/formic acid(900:100:1)

Injection volume: 20 μL

Gradient program:

TABLE 1 Times (min) Mobile phase A (vol %) Mobile phase B (vol %) 0 1000 10 100 0 40 35 65 40.1 0 100 50 0 100 50.1 100 0 60 100 0

The value of (X_(A)/Y)×100 is not particularly limited as long as it is4.4000 or less, and the smaller the value is, the more preferable it is.The value of (X_(A)/Y)×100 is preferably 4.0000 or less, more preferably3.5000 or less, and still more preferably 3.0000 or less. The lowerlimit of (X_(A)/Y)×100 is a detection limit. In terms of obtaining aneffect that matches an effort to decrease the value of (X_(A)/Y)×100(e.g., removal and separation of the peptide fragment group (A) bypurification), the value of (X_(A)/Y)×100 is preferably 0.0800 or more,more preferably 0.1000 or more, and still more preferably 0.1500 ormore.

The smaller the peak area value of the peptide fragment group (A), whichis calculated by an automatic integration method from an extracted ionchromatogram obtained by an LC-MS/MS analysis performed by using anaqueous solution prepared from the first composition and with analkaline phosphatase concentration of 10% by weight, is, the morepreferable it is. The peak area value of the peptide fragment group (A)is preferably 10,000 or less, more preferably 8,000 or less, and stillmore preferably 7,000 or less. The lower limit of the peak area value ofthe peptide fragment group (A) is a detection limit. In terms ofobtaining an effect that matches an effort to decrease the peak areavalue of the peptide fragment group (A) (e.g., removal and separation ofthe peptide fragment group (A) by purification), the peak area value ofthe peptide fragment group (A) is preferably 600 or more, morepreferably 800 or more, and still more preferably 1,000 or more.

The peak area value of an alkaline phosphatase, which is calculated byan automatic integration method from a chromatogram obtained by an LC-UVanalysis performed by using an aqueous solution prepared from the firstcomposition and with an alkaline phosphatase concentration of 10% byweight, is preferably 200,000 or more, more preferably 220,000 or more,and still more preferably 240,000 or more. The upper limit of the peakarea value of an alkaline phosphatase is not particularly limited. Thepeak area value of an alkaline phosphatase is preferably 500,000 orless, more preferably 400,000 or less, and still more preferably 350,000or less.

Content Ratio of Peptide Fragment Group (B) to Alkaline Phosphatase

In the second composition, the content ratio of the peptide fragmentgroup (B) to the alkaline phosphatase satisfies formula (B):

(X _(B) /Y)×100≤3.4000   (B).

In formula (B), X_(B) represents a peak area value of the peptidefragment group (B) calculated by an automatic integration method from anextracted ion chromatogram obtained by an LC-MS/MS analysis of thesecond composition, and Y represents a peak area value of the alkalinephosphatase calculated by an automatic integration method from achromatogram obtained by an LC-UV analysis of the second composition.The “peak area value of the peptide fragment group (B)” means a totalpeak area value of all peptide fragments constituting the peptidefragment group (B). The “peak area value of the alkaline phosphatase”means, when the second composition contains one alkaline phosphatase, apeak area value of the one alkaline phosphatase, and, when the secondcomposition contains two or more alkaline phosphatases, a total peakarea value of the two or more alkaline phosphatases (i.e., total peakarea value of all alkaline phosphatases contained in the secondcomposition). The descriptions on formula (A) (including thedescriptions on the LC-MS/MS analysis and the LC-UV analysis) also applyto formula (B) unless otherwise specified.

The m/z value of an ion of each peptide fragment constituting thepeptide fragment group (B) used to detect a peak of each peptidefragment constituting the peptide fragment group (B) is preferably 50 to2,200, more preferably 200 to 1,500, and still more preferably 300 to1,200.

The value of (X_(B)/Y)×100 is not particularly limited as long as it is3.4000 or less, and the smaller the value is, the more preferable it is.The value of (X_(B)/Y)×100 is preferably 3.0000 or less, more preferably2.8000 or less, and still more preferably 2.5000 or less. The lowerlimit of (X_(B)/Y)×100 is a detection limit. In terms of obtaining aneffect that matches an effort to decrease the value of (X_(B)/Y)×100(e.g., removal and separation of the peptide fragment group (B) bypurification), the value of (X_(B)/Y)×100 is preferably 0.0800 or more,more preferably 0.1000 or more, and still more preferably 0.1500 ormore.

The smaller the peak area value of the peptide fragment group (B), whichis calculated by an automatic integration method from an extracted ionchromatogram obtained by an LC-MS/MS analysis performed by using anaqueous solution prepared from the second composition and with analkaline phosphatase concentration of 10% by weight, is, the morepreferable it is. The peak area value of the peptide fragment group (B)is preferably 7,500 or less, more preferably 7,000 or less, and stillmore preferably 6,000 or less. The lower limit of the peak area value ofthe peptide fragment group (B) is a detection limit. In terms ofobtaining an effect that matches an effort to decrease the peak areavalue of the peptide fragment group (B) (e.g., removal and separation ofthe peptide fragment group (B) by purification), the peak area value ofthe peptide fragment group (B) is preferably 500 or more, morepreferably 600 or more, and still more preferably 800 or more.

The peak area value of an alkaline phosphatase, which is calculated byan automatic integration method from a chromatogram obtained by an LC-UVanalysis performed by using an aqueous solution prepared from the secondcomposition and with an alkaline phosphatase concentration of 10% byweight, is preferably 200,000 or more, more preferably 220,000 or more,and still more preferably 240,000 or more. The upper limit of the peakarea value of an alkaline phosphatase is not particularly limited. Thepeak area value of an alkaline phosphatase is preferably 500,000 orless, more preferably 400,000 or less, and still more preferably 350,000or less.

In an example in which the second composition a peptide fragmentconsisting of 5 to 50 consecutive amino acid residues selected frompositions 501 to 578 of the amino acid sequence set forth in SEQ ID NO:5 other than the peptide fragment group (B), the second composition mayor may not satisfy formula (A).

Content Ratio of Peptide Fragment Group (C) to Alkaline Phosphatase

In the third composition, the content ratio of the peptide fragmentgroup (C) to the alkaline phosphatase satisfies formula (C):

(X _(C) /Y)×100≤1.0000   (C).

In formula (C), X_(C) represents a peak area value of the peptidefragment group (C) calculated by an automatic integration method from anextracted ion chromatogram obtained by an LC-MS/MS analysis of the thirdcomposition, and Y represents a peak area value of the alkalinephosphatase calculated by an automatic integration method from achromatogram obtained by an LC-UV analysis of the third composition. The“peak area value of the peptide fragment group (C)” means a total peakarea value of all peptide fragments constituting the peptide fragmentgroup (C). The “peak area value of the alkaline phosphatase” means, whenthe third composition contains one alkaline phosphatase, a peak areavalue of the one alkaline phosphatase, and, when the third compositioncontains two or more alkaline phosphatases, a total peak area value ofthe two or more alkaline phosphatases (i.e., total peak area value ofall alkaline phosphatases contained in the third composition). Thedescriptions on formula (A) (including the descriptions on the LC-MS/MSanalysis and the LC-UV analysis) also apply to formula (C) unlessotherwise specified.

The m/z value of an ion of each peptide fragment constituting thepeptide fragment group (C) used to detect a peak of each peptidefragment constituting the peptide fragment group (C) is preferably 50 to2,200, more preferably 200 to 1,500, and still more preferably 300 to1,200.

The value of (X_(C)/Y)×100 is not particularly limited as long as it is1.0000 or less, and the smaller the value is, the more preferable it is.The value of (X_(C)/Y)×100 is preferably 0.9000 or less, more preferably0.8000 or less, and still more preferably 0.7000 or less. The lowerlimit of (X_(C)/Y)×100 is a detection limit. In terms of obtaining aneffect that matches an effort to decrease the value of (X_(C)/Y)×100(e.g., removal and separation of the peptide fragment group (C) bypurification), the value of (X_(C)/Y)×100 is preferably 0.0800 or more,more preferably 0.1000 or more, and still more preferably 0.1500 ormore.

The smaller the peak area value of the peptide fragment group (C), whichis calculated by an automatic integration method from an extracted ionchromatogram obtained by an LC-MS/MS analysis performed by using anaqueous solution prepared from the third composition and with analkaline phosphatase concentration of 10% by weight, is, the morepreferable it is. The peak area value of the peptide fragment group (C)is preferably 2,400 or less, more preferably 2,000 or less, and stillmore preferably 1,500 or less. The lower limit of the peak area value ofthe peptide fragment group (C) is a detection limit. In terms ofobtaining an effect that matches an effort to decrease the peak areavalue of the peptide fragment group (C) (e.g., removal and separation ofthe peptide fragment group (C) by purification), the peak area value ofthe peptide fragment group (C) is preferably 200 or more, morepreferably 300 or more, and still more preferably 400 or more.

The peak area value of an alkaline phosphatase, which is calculated byan automatic integration method from a chromatogram obtained by an LC-UVanalysis performed by using an aqueous solution prepared from the thirdcomposition and with an alkaline phosphatase concentration of 10% byweight, is preferably 200,000 or more, more preferably 220,000 or more,and still more preferably 240,000 or more. The upper limit of the peakarea value of an alkaline phosphatase is not particularly limited. Thepeak area value of an alkaline phosphatase is preferably 500,000 orless, more preferably 400,000 or less, and still more preferably 350,000or less.

In an example in which the third composition contains a peptide fragmentconsisting of 5 to 50 consecutive amino acid residues selected frompositions 501 to 578 of the amino acid sequence set forth in SEQ ID NO:5 other than the peptide fragment group (C), the third composition mayor may not satisfy formula (A).

Content Ratio of First Peptide Fragment to Alkaline Phosphatase

In an example in which the first, second, third, fourth, fifth or sixthcompositions contain the first peptide fragment, the content ratio ofthe first peptide fragment to the alkaline phosphatase preferablysatisfies formula (1):

(X ₁ /Y)×100≤1.0000   (1).

In formula (1), X₁ represents a peak area value of the first peptidefragment calculated by an automatic integration method from an extractedion chromatogram obtained by an LC-MS/MS analysis of the first, second,third, fourth, fifth or sixth composition, and Y represents a peak areavalue of the alkaline phosphatase calculated by an automatic integrationmethod from a chromatogram obtained by an LC-UV analysis of the first,second, third, fourth, fifth or sixth composition. The descriptions offormula (A) (including the descriptions on the LC-MS/MS analysis and theLC-UV analysis) also apply to formula (1) unless otherwise specified.

The value of (X₁/Y)×100 is not particularly limited as long as it is1.0000 or less, and the smaller the value is, the more preferable it is.The value of (X₁/Y)×100 is preferably 0.9000 or less, more preferably0.8000 or less, and still more preferably 0.7000 or less. The lowerlimit of (X₁/Y)×100 is a detection limit. In terms of obtaining aneffect that matches an effort to decrease the value of (X₁/Y)×100 (e.g.,removal and separation of the first peptide fragment by purification),the value of (X₁/Y)×100 is preferably 0.0500 or more, more preferably0.0600 or more, and still more preferably 0.0700 or more.

The smaller the peak area value of the first peptide fragment calculatedby an automatic integration method from an extracted ion chromatogramobtained by an LC-MS/MS analysis performed by using an aqueous solutionprepared from the first, second, third, fourth, fifth or sixthcompositions and with an alkaline phosphatase concentration of 10% byweight is, the more preferable it is. The peak area value of the firstpeptide fragment is preferably 3,000 or less, more preferably 2,500 orless, and still more preferably 2,000 or less. The lower limit of thepeak area value of the first peptide fragment is a detection limit. Interms of obtaining an effect that matches an effort to decrease the peakarea value of the first peptide fragment (e.g., removal and separationof the first peptide fragment by purification), the peak area value ofthe first peptide fragment is preferably 100 or more, more preferably150 or more, and still more preferably 200 or more.

The peak area value of an alkaline phosphatase calculated by anautomatic integration method from a chromatogram obtained by an LC-UVanalysis performed by using an aqueous solution prepared from the first,second, third, fourth, fifth or sixth compositions and with an alkalinephosphatase concentration of 10% by weight is preferably 200,000 ormore, more preferably 220,000 or more, and still more preferably 240,000or more. The upper limit of the peak area value of an alkalinephosphatase is not particularly limited. The peak area value of analkaline phosphatase is preferably 500,000 or less, more preferably400,000 or less, and still more preferably 350,000 or less.

Content Ratio of Second Peptide Fragment to Alkaline Phosphatase

In the fourth composition, the content ratio of the second peptidefragment to the alkaline phosphatase satisfies formula (2):

(X ₂ /Y)×100≤1.6000   (2).

In an example in which the first, second, third, fifth or sixthcompositions contain the second peptide fragment, the content ratio ofthe second peptide fragment to the alkaline phosphatase preferablysatisfies formula (2):

(X ₂ /Y)×100≤1.6000   (2).

In formula (2), X₂ represents a peak area value of the second peptidefragment calculated by an automatic integration method from an extractedion chromatogram obtained by an LC-MS/MS analysis of the first, second,third, fourth, fifth or sixth compositions, and Y represents a peak areavalue of the alkaline phosphatase calculated by an automatic integrationmethod from a chromatogram obtained by an LC-UV analysis of the first,second, third, fourth, fifth or sixth compositions. The descriptions onformula (A) (including the descriptions on the LC-MS/MS analysis and theLC-UV analysis) also apply to formula (2) unless otherwise specified.

The value of (X₂/Y)×100 is not particularly limited as long as it is1.6000 or less, and the smaller the value is, the more preferable it is.The value of (X₂/Y)×100 is preferably 1.5000 or less, more preferably1.2000 or less, and still more preferably 1.0000 or less. The lowerlimit of (X₂/Y)×100 is a detection limit. In terms of obtaining aneffect that matches an effort to decrease the value of (X₂/Y)×100 (e.g.,removal and separation of the second peptide fragment by purification),the value of (X₂/Y)×100 is preferably 0.0500 or more, more preferably0.0600 or more, and still more preferably 0.0700 or more.

The smaller the peak area value of the second peptide fragmentcalculated by an automatic integration method from an extracted ionchromatogram obtained by an LC-MS/MS analysis performed by using anaqueous solution prepared from the first, second, third, fourth, fifthor sixth compositions and with an alkaline phosphatase concentration of10% by weight is, the more preferable it is. The peak area value of thesecond peptide fragment is preferably 4,500 or less, more preferably3,000 or less, and still more preferably 2,500 or less. The lower limitof the peak area value of the second peptide fragment is a detectionlimit. In terms of obtaining an effect that matches an effort todecrease the peak area value of the second peptide fragment (e.g.,removal and separation of the second peptide fragment by purification),the peak area value of the second peptide fragment is preferably 100 ormore, more preferably 150 or more, and still more preferably 200 ormore.

The peak area value of an alkaline phosphatase calculated by anautomatic integration method from a chromatogram obtained by an LC-UVanalysis performed by using an aqueous solution prepared from the first,second, third, fourth, fifth or sixth compositions and with an alkalinephosphatase concentration of 10% by weight is preferably 200,000 ormore, more preferably 220,000 or more, and still more preferably 240,000or more. The upper limit of the peak area value of an alkalinephosphatase is not particularly limited. The peak area value of analkaline phosphatase is preferably 500,000 or less, more preferably400,000 or less, and still more preferably 350,000 or less.

Content Ratio of Third Peptide Fragment to Alkaline Phosphatase

In the fifth composition, the content ratio of the third peptidefragment to the alkaline phosphatase satisfies formula (3):

(X ₃ /Y)×100≤0.2000   (3).

In an example in which the first, second, third, fourth or sixthcompositions contain the third peptide fragment, the content ratio ofthe third peptide fragment to the alkaline phosphatase preferablysatisfies formula (3):

(X ₃ /Y)×100≤0.2000   (3).

In formula (3), X₃ represents a peak area value of the third peptidefragment calculated by an automatic integration method from an extractedion chromatogram obtained by an LC-MS/MS analysis of the first, second,third, fourth, fifth or sixth compositions, and Y represents a peak areavalue of the alkaline phosphatase calculated by an automatic integrationmethod from a chromatogram obtained by an LC-UV analysis of the first,second, third, fourth, fifth or sixth compositions. The descriptions offormula (A) (including the descriptions on the LC-MS/MS analysis and theLC-UV analysis) also apply to formula (3) unless otherwise specified.

The value of (X₃/Y)×100 is not particularly limited as long as it is0.2000 or less, and the smaller the value is, the more preferable it is.The value of (X₃/Y)×100 is preferably 0.1800 or less, more preferably0.1700 or less, and still more preferably 0.1500 or less. The lowerlimit of (X₃/Y)×100 is a detection limit. In terms of obtaining aneffect that matches an effort to decrease the value of (X₃/Y)×100 (e.g.,removal and separation of the third peptide fragment by purification),the value of (X₃/Y)×100 is preferably 0.0500 or more, more preferably0.0600 or more, and still more preferably 0.0700 or more.

The smaller the peak area value of the third peptide fragment calculatedby an automatic integration method from an extracted ion chromatogramobtained by an LC-MS/MS analysis performed by using an aqueous solutionprepared from the first, second, third, fourth, fifth or sixthcompositions and with an alkaline phosphatase concentration of 10% byweight is, the more preferable it is. The peak area value of the thirdpeptide fragment is preferably 500 or less, more preferably 450 or less,and still more preferably 400 or less. The lower limit of the peak areavalue of the third peptide fragment is a detection limit. In terms ofobtaining an effect that matches an effort to decrease the peak areavalue of the third peptide fragment (e.g., removal and separation of thethird peptide fragment by purification), the peak area value of thethird peptide fragment is preferably 100 or more, more preferably 150 ormore, and still more preferably 200 or more.

The peak area value of an alkaline phosphatase calculated by anautomatic integration method from a chromatogram obtained by an LC-UVanalysis performed by using an aqueous solution prepared from the first,second, third, fourth, fifth or sixth compositions and with an alkalinephosphatase concentration of 10% by weight is preferably 200,000 ormore, more preferably 220,000 or more, and still more preferably 240,000or more. The upper limit of the peak area value of an alkalinephosphatase is not particularly limited. The peak area value of analkaline phosphatase is preferably 500,000 or less, more preferably400,000 or less, and still more preferably 350,000 or less.

Content Ratio of Fourth Peptide Fragment to Alkaline Phosphatase

In the sixth composition, the content ratio of the fourth peptidefragment to the alkaline phosphatase satisfies formula (4):

(X ₄ /Y)×100≤0.3500   (4).

In an example in which the first, second, third, fourth or fifthcompositions contain the fourth peptide fragment, the content ratio ofthe fourth peptide fragment to the alkaline phosphatase preferablysatisfies formula (4):

(X ₄ /Y)×100≤0.3500   (4).

In formula (4), X₄ represents a peak area value of the fourth peptidefragment calculated by an automatic integration method from an extractedion chromatogram obtained by an LC-MS/MS analysis of the first, second,third, fourth, fifth or sixth compositions, and Y represents a peak areavalue of the alkaline phosphatase calculated by an automatic integrationmethod from a chromatogram obtained by an LC-UV analysis of the first,second, third, fourth, fifth or sixth compositions. The descriptions offormula (A) (including the descriptions on the LC-MS/MS analysis and theLC-UV analysis) also apply to formula (4) unless otherwise specified.

The value of (X₄/Y)×100 is not particularly limited as long as it is0.3500 or less, and the smaller the value is, the more preferable it is.The value of (X₄/Y)×100 is preferably 0.3200 or less, more preferably0.3000 or less, and still more preferably 0.2800 or less. The lowerlimit of (X₄/Y)×100 is a detection limit. In terms of obtaining aneffect that matches an effort to decrease the value of (X₄/Y)×100 (e.g.,removal and separation of the fourth peptide fragment by purification),the value of (X₄/Y)×100 is preferably 0.0800 or more, more preferably0.1000 or more, and still more preferably 0.1500 or more.

The smaller the peak area value of the fourth peptide fragmentcalculated by an automatic integration method from an extracted ionchromatogram obtained by an LC-MS/MS analysis performed by using anaqueous solution prepared from the first, second, third, fourth, fifthor sixth compositions and with an alkaline phosphatase concentration of10% by weight is, the more preferable it is. The peak area value of thefourth peptide fragment is preferably 1000 or less, more preferably 900or less, and still more preferably 800 or less. The lower limit of thepeak area value of the fourth peptide fragment is a detection limit. Interms of obtaining an effect that matches an effort to decrease the peakarea value of the fourth peptide fragment (e.g., removal and separationof the fourth peptide fragment by purification), the peak area value ofthe fourth peptide fragment is preferably 100 or more, more preferably150 or more, and still more preferably 200 or more.

The peak area value of an alkaline phosphatase calculated by anautomatic integration method from a chromatogram obtained by an LC-UVanalysis performed by using an aqueous solution prepared from the first,second, third, fourth, fifth or sixth compositions and with an alkalinephosphatase concentration of 10% by weight is preferably 200,000 ormore, more preferably 220,000 or more, and still more preferably 240,000or more. The upper limit of the peak area value of an alkalinephosphatase is not particularly limited. The peak area value of analkaline phosphatase is preferably 500,000 or less, more preferably400,000 or less, and still more preferably 350,000 or less.

Alkaline Phosphatase Specific Activity

Our compositions preferably have an alkaline phosphatase specificactivity of 2,000 U/mg or more. The alkaline phosphatase specificactivity of the compositions is more preferably 2,500 U/mg or more, andstill more preferably 3,000 U/mg or more. The alkaline phosphatasespecific activity of the compositions is measured as follows. Bymeasuring the absorbance at 405 nm derived from p-nitrophenol producedby adding an alkaline phosphatase to an aqueous solution ofp-nitrophenylphosphate, it is possible to calculate the specificactivity of the alkaline phosphatase.

Other Components

Our compositions can contain one or two or more other components.Examples of the other components include aqueous vehicles such as water,metal salts such as a magnesium salt and a sodium salt, surfactants,organic acids, glycerin and the like.

Our compositions can be used for various applications requiring analkaline phosphatase activity, and can contain one or two or more othercomponents selected according to the applications.

In one example, the composition contains a protein such as an antigenand an antibody. In this example, the composition can be used forlabeling a protein such as an antigen and an antibody with the alkalinephosphatase. In other words, in one example, the composition is acomposition used to label a protein with the alkaline phosphatase.Labeling of a protein with the alkaline phosphatase can be performed byreacting a succinimide ester of the alkaline phosphatase, which isobtained by esterifying a carboxyl group of the alkaline phosphatasewith succinimide, with an amino group of the protein.

In one example, the composition contains a substrate for the alkalinephosphatase. In this example, the composition can be used todephosphorylate a substrate for the alkaline phosphatase. In otherwords, in one example, the composition is used to dephosphorylate asubstrate for the alkaline phosphatase. The substrate for the alkalinephosphatase is not particularly limited as long as the substrate is acompound having a phosphoric monoester bond. Examples of the substratefor the alkaline phosphatase include a nucleic acid, a phospholipid,pyrophosphoric acid and the like. When the substrate for the alkalinephosphatase is treated with the composition, the phosphoric monoesterbond of the substrate for the alkaline phosphatase is hydrolyzed by thealkaline phosphatase, resulting in dephosphorylation of the substratefor the alkaline phosphatase.

Preferably, the composition contains a nucleic acid. In this example,the composition can be used to dephosphorylate a nucleic acid. In otherwords, preferably, the composition is used to dephosphorylate a nucleicacid. The peptide fragment group (A), the peptide fragment group (B),the peptide fragment group (C), the second peptide fragment, the thirdpeptide fragment or the fourth peptide fragment coexisting in thealkaline phosphatase has a possibility of adversely influencing when thenucleic acid is dephosphorylated by the alkaline phosphatase. In thisregard, in the composition, the content ratio of the peptide fragmentgroup (A), the peptide fragment group (B), the peptide fragment group(C), the second peptide fragment, the third peptide fragment or thefourth peptide fragment to the alkaline phosphatase satisfies the abovepredetermined formula. In other words, in the composition, the relativecontent of the peptide fragment group (A), the peptide fragment group(B), the peptide fragment group (C), the second peptide fragment, thethird peptide fragment or the fourth peptide fragment has been reduced.Therefore, by using the composition, it is possible to reduce theadverse influence of the peptide fragment group (A), the peptidefragment group (B), the peptide fragment group (C), the second peptidefragment, the third peptide fragment or the fourth peptide fragment thatcan occur when the nucleic acid is dephosphorylated by the alkalinephosphatase, thus enabling improvement in the dephosphorylationefficiency of the nucleic acid. By treating the nucleic acid with thecomposition, the 5′ end and/or the 3′ end of the nucleic acid can bedephosphorylated. By dephosphorylating the 5′ end and/or the 3′ end ofthe nucleic acid, it is possible to improve the labeling efficiency whenthe 5′ end and/or the 3′ end of the nucleic acid is/are labeled with thelabeling substance. Particularly, when ³²P is used as the labelingsubstance, this effect is remarkable. By dephosphorylating the 5′ endand/or the 3′ end of a vector used for DNA cloning, self-ligation of thevector can be prevented, thus enabling a reduction in the background ofa transformed cell.

Examples of the nucleic acid include nucleic acids such as DNA, RNA,peptide nucleic acid (PNA) and locked nucleic acid (LNA) or a nucleicacid derivative. Examples of the nucleic acid derivative include anucleic acid derivative containing a modified nucleotide (e.g., anucleotide that has undergone reconstitution of a nucleotide or basecontaining a halogen group, an alkyl group such as a methyl group, analkoxy group such as a methoxy group, a thio group and a carboxymethylgroup and the like, saturation of a double bond, deamination,substitution of an oxygen molecule with a sulfur molecule and the like).The nucleic acid may be single-stranded or double-stranded. Examples ofthe DNA include chromosomal DNA, viral DNA, DNA of a bacterium, a fungusand the like, cDNA, fragments thereof and the like. Examples of the RNAinclude mRNA, rRNA, small RNA, fragments thereof and the like. Thenucleic acid may be chemically synthesized DNA, RNA and the like.Specific examples of the nucleic acid include a gene of a pathogen, avirus and the like, or a part thereof, a causative gene for geneticdisease or a part thereof and the like.

The nucleic acid can be prepared by an extraction by a conventionalmethod from, for example, a biomaterial, a virus, a bacterium, a fungus,a food and drink and the like. Examples of the biomaterial include bodyfluids such as blood, serum, plasma, urine, stool, spinal fluid, saliva,swab and tissue fluid, a cell, a tissue and the like. The biomaterialmay be animal-derived or plant-derived.

The amount of the nucleic acid contained in the composition can beappropriately adjusted according to the intended use of the composition(e.g., detection of the target nucleic acid) and the like. For example,when a certain nucleic acid (i.e., a target nucleic acid) among nucleicacids contained in the composition is intended to be detected, it ispossible to amplify the target nucleic acid by performing a nucleic acidamplification method such as PCR, by using the nucleic acids containedin the composition as a template. This enables improvement in thedetection sensitivity of the target nucleic acid.

The length (number of bases) of the nucleic acid can be appropriatelyadjusted according to the intended use of the composition (e.g.,detection of the target nucleic acid) or the like. For example, when thenucleic acid is intended to be detected, the length of the nucleic acidis usually 10 to 300 bases, preferably 10 to 100 bases, and morepreferably 15 to 100 bases. The length of the nucleic acid can beappropriately adjusted by fragmentation treatment. The length of thenucleic acid is, for example, a length at which the nucleic acid can behybridized with a probe. When the nucleic acid is long (e.g., 1,500bases or more, particularly 4,000 bases or more), it is preferable toperform fragmentation treatment of the nucleic acid and to adjust thelength of the nucleic acid to an appropriate length. When fragmentationtreatment is performed, it is not necessarily that a specific nucleicacid fragment is selected from the generated nucleic acid fragments, andthe fragmentation product can be used as it is.

Examples of a method of cleaving the nucleic acid for fragmentationinclude a method of cleaving by irradiation with ultrasonic waves, amethod of cleaving with an enzyme, a method of cleaving with arestriction enzyme, a method using a nebulizer, a method of cleavingwith an acid or an alkali and the like. In the method of cleaving withultrasonic waves, by controlling the output intensity and theirradiation time of the ultrasonic waves with which the nucleic acid isirradiated, it is possible to cleavage the nucleic acid into a desiredlength.

Preferably, the composition contains a dephosphorylated nucleic acid.The descriptions on the nucleic acid are the same as mentioned above.The dephosphorylated nucleic acid has a 5′ end and/or a 3′ end, each ofwhich has been dephosphorylated by the alkaline phosphatase. In thisexample, the composition can be used to prepare a labeled nucleic acidcontaining the dephosphorylated nucleic acid and a labeling substancebound to the dephosphorylated nucleic acid. In other words, preferably,the composition is a composition used to prepare a labeled nucleic acidcontaining the dephosphorylated nucleic acid and a labeling substancebound to the dephosphorylated nucleic acid. The peptide fragment group(A), the peptide fragment group (B), the peptide fragment group (C), thesecond peptide fragment, the third peptide fragment or the fourthpeptide fragment coexisting in the alkaline phosphatase has apossibility of adversely influencing when the nucleic acid isdephosphorylated by the alkaline phosphatase and/or when the labelingsubstance is bound to the dephosphorylated nucleic acid. In this regard,in the composition, the content ratio of the peptide fragment group (A),the peptide fragment group (B), the peptide fragment group (C), thesecond peptide fragment, the third peptide fragment or the fourthpeptide fragment to the alkaline phosphatase satisfies the abovepredetermined formula. In other words, in the composition, the relativecontents of the peptide fragment group (A), the peptide fragment group(B), the peptide fragment group (C), the second peptide fragment, thethird peptide fragment or the fourth peptide fragment has been reduced.Therefore, by using the composition, it is possible to reduce theadverse influence of the peptide fragment group (A), the peptidefragment group (B), the peptide fragment group (C), the second peptidefragment, the third peptide fragment or the fourth peptide fragment thatcan occur when the nucleic acid is dephosphorylated by the alkalinephosphatase and/or when the labeling substance is bound to thedephosphorylated nucleic acid, thus enabling improvement in thedephosphorylation efficiency of the nucleic acid and/or the labelingefficiency of the dephosphorylated nucleic acid.

Preferably, the composition contains a labeled nucleic acid containing adephosphorylated nucleic acid and a labeling substance bound to thedephosphorylated nucleic acid. The descriptions on the nucleic acid arethe same as mentioned above. The dephosphorylated nucleic acid has a 5′end and/or a 3′ end, each of which has been dephosphorylated by thealkaline phosphatase. The labeling substance is bound to the 5′ endand/or the 3′ end of the dephosphorylated nucleic acid.

As the labeling substance, for example, a fluorescent dye, a fluorescentprotein, a chemiluminescent body, a metal complex, a metal fineparticle, biotin, a radioisotope or the like can be used. The reactionconditions when the target nucleic acid is labeled can be appropriatelyadjusted according to the type of the labeling substance. When thelabeling substance is a fluorescent dye, the fluorescent dye can bedetected by using a fluorescence microscope, a fluorescence scanner andthe like.

Examples of the fluorescent dye include organic fluorescent dyes such asa fluorescein-based dye, a rhodamine-based dye, an Alexa Fluor(manufactured by Invitrogen)-based dye, a BODIPY (manufactured byInvitrogen)-based dye, a cascade-based dye, a coumarin-based dye, aneosin-based dye, an NBD-based dye, a pyrene-based dye, a Texas Red-baseddye and a cyanine-based dye.

Specific examples of the organic fluorescent dye include5-carboxy-fluorescein, 6-carboxy-fluorescein, 5,6-dicarboxy-fluorescein,6-carboxy-2′,4,4′,5′,7,7′-hexachloro-fluorescein,6-carboxy-2′,4,7,7′-tetrachloro-fluorescein,6-carboxy-4′,5′-dichloro-2′,7′-dimethoxy-fluorescein,naphtho-fluorescein, 5-carboxy-rhodamine, 6-carboxy-rhodamine,5,6-dicarboxy-rhodamine, rhodamine 6G, tetramethylrhodamine,X-rhodamine, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, AlexaFluor 488, Alexa Fluor 500, Alexa Fluor 514, Alexa Fluor 532, AlexaFluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, AlexaFluor 610, Alexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647, AlexaFluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, BODIPY FL,BODIPY TMR, BODIPY 493/503, BODIPY 530/550, BODIPY 558/568, BODIPY564/570, BODIPY 576/589, BODIPY 581/591, BODIPY 630/650, BODIPY 650/665(all of which are manufactured by Invitrogen), methoxycoumarin, eosin,NBD, pyrene, Cy5, Cy5.5, Cy7 and the like.

In the example in which the composition contains a labeled nucleic acidcontaining a dephosphorylated nucleic acid and a labeling substancebound to the dephosphorylated nucleic acid, the composition can be usedas a nucleic acid sample to be subjected to a nucleic acid detectionmethod. In other words, preferably, the composition is a nucleic acidsample to be subjected to the nucleic acid detection method. The labelednucleic acid can contain a target nucleic acid to be detected and anucleic acid other than the target nucleic acid. In the nucleic aciddetection method, the target nucleic acid contained in the nucleic acidsample can be detected by using the labeling substance of the targetnucleic acid as an index. The nucleic acid detection method is notparticularly limited, and can be appropriately selected from knownnucleic acid detection methods. The target nucleic acid can be detected,for example, by using the hybridization method. In one example of thehybridization method, the target nucleic acid can be detected by using aprobe that can be hybridized with the target nucleic acid. In oneexample of the nucleic acid detection method using a probe, the probe isbrought into contact with the nucleic acid sample containing the targetnucleic acid to hybridize the probe with the target nucleic acid, andthe target nucleic acid hybridized with the probe can be detected byusing the labeling substance of the target nucleic acid as an index.When a nucleic acid other than the target nucleic acid is contained inthe sample, it is preferable that, after the target nucleic acid isbrought into contact with the probe, the nucleic acid that was nothybridized with the probe is removed by washing or the like.

The reaction conditions when the target nucleic acid is hybridized withthe probe can be appropriately adjusted according to chain length of thetarget nucleic acid, the chain length of the probe and the like. Thereaction time is usually 30 to 1,200 minutes, and preferably 60 to 360minutes. The reaction temperature is usually 25 to 60° C., andpreferably 30 to 40° C. The reaction is usually performed in an aqueousvehicle such as water.

The amount of the target nucleic acid or probe used is not particularlylimited as long as the hybridization between the target nucleic acid andthe probe can occur and the labeling substance bound to the targetnucleic acid can be detected, and the amount can be appropriatelyadjusted according to the chain length of the target nucleic acid, thechain length of the probe, the type of the labeling substance and thelike.

As the probe, for example, nucleic acids such as DNA, RNA, peptidenucleic acid (PNA) and locked nucleic acid (LNA) or a nucleic acidderivative can be used. Examples of the nucleic acid derivative includea nucleic acid derivative containing a modified nucleotide (e.g., anucleotide that has undergone reconstitution of a nucleotide or basecontaining a halogen group, an alkyl group such as a methyl group, analkoxy group such as a methoxy group, a thio group and a carboxymethylgroup and the like, saturation of a double bond, deamination,substitution of an oxygen molecule with a sulfur molecule and the like).

The probe has a base sequence complementary to at least a part of thebase sequence of the target nucleic acid, and can be hybridized with thetarget nucleic acid. When the target nucleic acid is double-stranded,the probe may be hybridized with a sense strand or may be hybridizedwith an antisense strand. The base sequence of the probe may becomplementary to any part of the base sequence of the target nucleicacid, and is preferably complementary to a highly-specific part of thebase sequence of the target nucleic acid. In other words, the basesequence of the probe is preferably complementary to a base sequencewhich other nucleic acids contained in the sample do not have, of thebase sequence of the target nucleic acid.

Of the base sequence of the probe, the length (number of bases) of thepart complementary to the base sequence of the target nucleic acid isnot particularly limited, and is usually 10 to 150 bases, preferably 20to 100 bases, and more preferably 20 to 70 bases. The probe may becomposed of a base sequence complementary to the base sequence of thetarget nucleic acid, or may include a base sequence not complementary tothe base sequence of the target nucleic acid. The full length (totalnumber of bases) of the probe is usually 10 to 300 bases, preferably 20to 200 bases, and more preferably 15 to 100 bases.

The probe may be any of a commercially available product, a syntheticproduct, a prepared product from a living body and the like. A nucleicacid having a length of up to 200 bases, which is referred to as anoligonucleic acid, can be easily artificially synthesized with asynthesizer.

The probe is preferably fixed to a support. In other words, preferably,the nucleic acid detection method is a nucleic acid detection methodusing a nucleic acid microarray. The nucleic acid microarray has asupport and a plurality of probes fixed to the surface of the support.In the nucleic acid detection method using a nucleic acid microarray,the labeled target nucleic acid is brought into contact with a nucleicacid microarray provided with a probe that can be hybridized with thetarget nucleic acid, and the target nucleic acid hybridized with theprobe can be detected by using the labeling substance bound to thetarget nucleic acid as an index. When a nucleic acid other than thetarget nucleic acid is contained in the sample, it is preferable that,after the target nucleic acid is brought into contact with the nucleicacid microarray, the nucleic acid that has not been hybridized with theprobe on the nucleic acid microarray is removed by washing or the like.By using a nucleic acid microarray provided with a plurality of probes,two or more target nucleic acids can be simultaneously detected.

The support is not particularly limited as long as it can fix the probe.Examples of the support include a slide, a membrane, a bead and thelike. Examples of the material of the support include inorganicmaterials such as glass, ceramic and silicon, and polymers such aspolyethylene terephthalate, cellulose acetate, polycarbonate,polystyrene, polymethyl methacrylate and silicone rubber and the like.

Fixation of the probe to the support can be performed in accordance witha conventional method. As a method of fixing the probe to the support, amethod of synthesizing an oligonucleic acid on the top surface of thesupport, a method of adding dropwise an oligonucleic acid synthesized inadvance to the top surface of the support to fix and the like are known.Examples of the former method include the method by U.S. Pat. No.5,705,610 A, the method by U.S. Pat. No. 6,142,266 A, the method by U.S.Pat. No. 7,037,659 A and the like. In those methods, since an organicsolvent is used during DNA synthesis reaction, the support is desirablya material that is resistance to an organic solvent. For example, it ispossible to use a glass support having an irregular structure fabricatedby using the method disclosed in JP H10-503841 A. Particularly, in themethod by U.S. '659, since the back of the support is irradiated withlight to control DNA synthesis, the support is preferably a materialhaving translucency. Examples of the latter method include the method byJP 3922454 B2, a method using a glass capillary and the like. As anexample of the glass capillary, it is possible to use a self-made glasscapillary, commercially available products such as a micropipette(manufactured by Micro Support Co., Ltd.; MP-005) and the like.

As a method of detecting the target nucleic acid, the sandwichhybridization method can be used. In the sandwich hybridization method,a first probe (capture probe) fixed to the support and a second probe(detection probe) not fixed to the support are used. The capture probeand the detection probe each have a base sequence complementary todifferent parts of the target nucleic acid, and can be hybridized withdifferent parts of the target nucleic acid. The target nucleic acid ishybridized with the detection probe and the capture probe, thus forminga complex. By detecting a labeling substance contained in this complex,the target nucleic acid can be detected.

The sequence identity between the base sequence of the detection probeand the base sequence of the capture probe is preferably low. Thesequence identity is preferably 20% or less, and more preferably 10% orless. In this regard, the identity between two base sequences is anumerical value obtained by aligning two sequences (inserting a gap, ifnecessary) so that bases are matched as many as possible, and then bydividing the number of matched bases by total number of bases (thehigher number of bases when the number of bases of two base sequences isdifferent), and the identity can be easily calculated with commerciallyavailable software such as FASTA and BLAST (also provided via theinternet).

The signal detected in the method of detecting the target nucleic acid(e.g., intensity of the detected labeling substance) is compared with asurrounding noise. Specifically, the signal value obtained from aposition on the support at which a probe is fixed is compared with thesignal value (noise value) obtained from a position of the support atwhich no probe is fixed, and a ratio of the former numerical value tothe noise value is defined as an S/N ratio. The detection accuracy canbe represented by the S/N ratio. In other words, the larger the S/Nratio is, the higher the detection accuracy is, and the smaller the S/Nratio is, the lower the detection accuracy is.

In the example in which the composition contains a labeled nucleic acidcontaining a dephosphorylated nucleic acid and a labeling substancebound to the dephosphorylated nucleic acid, by using the composition asa nucleic acid sample in the nucleic acid detection method, it ispossible to improve the detection sensitivity of the target nucleicacid. This effect is remarkable in a nucleic acid detection method usingan extremely small amount (preferably 5 to 1,000 μL, more preferably 5to 500 μL) of a nucleic acid sample. In the nucleic acid detectionmethod using an extremely small amount of a nucleic acid sample, thepeptide fragment group (A), the peptide fragment group (B), the peptidefragment group (C), the second peptide fragment, the third peptidefragment or the fourth peptide fragment contained in the nucleic acidsample has a possibility of adversely influencing the detectionsensitivity. In this regard, in the composition, the content ratio ofthe peptide fragment group (A), the peptide fragment group (B), thepeptide fragment group (C), the second peptide fragment, the thirdpeptide fragment or the fourth peptide fragment to the alkalinephosphatase satisfies the above predetermined formula. In other words,in the composition, the relative content of the peptide fragment group(A), the peptide fragment group (B), the peptide fragment group (C), thesecond peptide fragment, the third peptide fragment or the fourthpeptide fragment has been reduced. Therefore, in the nucleic aciddetection method using an extremely small amount of a nucleic acidsample, by using the composition as the nucleic acid sample, it ispossible to reduce the adverse influence of the peptide fragment group(A), the peptide fragment group (B), the peptide fragment group (C), thesecond peptide fragment, the third peptide fragment or the fourthpeptide fragment, thus enabling remarkable improvement in the detectionsensitivity of the target nucleic acid.

Preferably, the nucleic acid detection method is a nucleic aciddetection method using a nucleic acid microarray. In the nucleic aciddetection method using a nucleic acid microarray, an extremely smallamount (preferably 5 to 1,000 μL, more preferably 5 to 500 μL) of anucleic acid sample is used. Therefore, in the nucleic acid detectionmethod using a nucleic acid microarray, by using the composition as thenucleic acid sample, it is possible to reduce the adverse influence ofthe peptide fragment group (A), the peptide fragment group (B), thepeptide fragment group (C), the second peptide fragment, the thirdpeptide fragment or the fourth peptide fragment, thus enablingremarkable improvement in the detection sensitivity of the targetnucleic acid.

Production Method

The composition can be produced by separating the peptide fragment group(A), the peptide fragment group (B), the peptide fragment group (C), thesecond peptide fragment, the third peptide fragment or the fourthpeptide fragment from an alkaline phosphatase extract from an organ of abovine, a shrimp or the like, an alkaline phosphatase extract from amicroorganism into which a gene encoding an alkaline phosphatase hasbeen introduced, a bacterial cell homogenate of a microorganism intowhich a gene encoding an alkaline phosphatase has been introduced, acommercially available alkaline phosphatase product and the like.Examples of the method of separating the peptide fragment group (A), thepeptide fragment group (B), the peptide fragment group (C), the secondpeptide fragment, the third peptide fragment or the fourth peptidefragment include dialysis, salting out, gel filtration, ultrafiltration,membrane separation, ion exchange, column chromatography,electrophoresis and the like. Regarding the method of separating thepeptide fragment, one separation method may be used alone, or two ormore separation methods may be used in combination. For example, bypurifying a commercially available alkaline phosphatase product bycolumn chromatography or the like, it is possible to make the content ofthe peptide fragment group (A), the peptide fragment group (B), thepeptide fragment group (C), the second peptide fragment, the thirdpeptide fragment or the fourth peptide fragment to the alkalinephosphatase be to a desired range. The column chromatography is, forexample, liquid chromatography. The column and the mobile phase used inliquid chromatography is not particularly limited as long as the peptidefragment group (A), the peptide fragment group (B), the peptide fragmentgroup (C), the second peptide fragment, the third peptide fragment orthe fourth peptide fragment can be separated, and it is preferable touse a C18-supported reverse-phase column.

Use

The composition can be used for various methods requiring an alkalinephosphatase activity.

In one example, the composition is used for a method of producing adephosphorylated nucleic acid, the method including the steps of:

providing the composition;

providing a nucleic acid; and

treating the nucleic acid with the composition to dephosphorylate thenucleic acid. The peptide fragment group (A), the peptide fragment group(B), the peptide fragment group (C), the second peptide fragment, thethird peptide fragment or the fourth peptide fragment coexisting in thealkaline phosphatase has a possibility of adversely influencing when thenucleic acid is dephosphorylated by the alkaline phosphatase. In thisregard, in the composition, the content ratio of the peptide fragmentgroup (A), the peptide fragment group (B), the peptide fragment group(C), the second peptide fragment, the third peptide fragment or thefourth peptide fragment to the alkaline phosphatase satisfies the abovepredetermined formula. In other words, in the composition, the relativecontent of the peptide fragment group (A), the peptide fragment group(B), the peptide fragment group (C), the second peptide fragment, thethird peptide fragment or the fourth peptide fragment has been reduced.Thus, by treating the nucleic acid with the composition, it is possibleto improve the dephosphorylation efficiency of the nucleic acid.

In one example, the composition is used for a method of producing alabeled nucleic acid, the method including the steps of:

providing the composition;

providing a nucleic acid;

providing a labeling substance;

treating the nucleic acid with the composition to dephosphorylate thenucleic acid; and

binding the labeling substance to the dephosphorylated nucleic acid. Thepeptide fragment group (A), the peptide fragment group (B), the peptidefragment group (C), the second peptide fragment, the third peptidefragment or the fourth peptide fragment coexisting in the alkalinephosphatase has a possibility of adversely influencing when the nucleicacid is dephosphorylated by the alkaline phosphatase and/or when thelabeling substance is bound to the dephosphorylated nucleic acid. Inthis regard, in the composition, the content ratio of the peptidefragment group (A), the peptide fragment group (B), the peptide fragmentgroup (C), the second peptide fragment, the third peptide fragment orthe fourth peptide fragment to the alkaline phosphatase satisfies theabove predetermined formula. In other words, in the composition, therelative content of the peptide fragment group (A), the peptide fragmentgroup (B), the peptide fragment group (C), the second peptide fragment,the third peptide fragment or the fourth peptide fragment has beenreduced. Thus, by treating the nucleic acid with the composition, it ispossible to improve the dephosphorylation efficiency of the nucleic acidand/or the labeling efficiency of the dephosphorylated nucleic acid.

In the step of treating the nucleic acid with the composition todephosphorylate the nucleic acid, the reaction conditions can beappropriately adjusted. The reaction time is usually 10 to 60 minutes,and preferably 20 to 50 minutes. The reaction temperature is usually 20to 60° C., and preferably 25 to 45° C. The reaction is usually performedin an aqueous vehicle such as water. The nucleic acid is, for example,DNA, RNA and the like. When the nucleic acid is treated with thecomposition, the 5′ end and/or the 3′ end of the nucleic acid isdephosphorylated.

In the step of binding the labeling substance to the dephosphorylatednucleic acid, as the labeling substance, for example, a fluorescent dye,a fluorescent protein, a chemiluminescent body, a metal complex, a metalfine particle, biotin, a radioisotope, and the like can be used. Thereaction conditions can be appropriately adjusted according to the typeof the labeling substance. The dephosphorylated nucleic acid has a 5′end and/or a 3′ end, each of which has been dephosphorylated by thealkaline phosphatase, and the labeling substance can be bound to thedephosphorylated 5′ end and/or 3′ end.

EXAMPLES

Our compositions and methods will be described in detail by way ofExamples, but this disclosure is not limited to the following Examples.

Conditions of LC-MS/MS Analysis

Conditions of the LC-MS/MS analysis used in Examples and ComparativeExamples were as follows.

Apparatus Configuration

Mass spectrometer: maXis impact (manufactured by Bruker Daltnics, Inc.)

Conditions of Mass Spectrometry

Ionization method: ESI

Measured ion: cation

Capillary voltage: 4,500 V

Nebulizer: 2.0 bar

Dry gas: 8.0 L/min

Detector voltage: 1,823 V

Measuring span (MS): m/z 50 to 2,200

MS/MS Conditions

Measuring span (MS): m/z 50 to 2,200

Collision gas: nitrogen

Conditions of LC-UV Analysis

Conditions of the LC-UV analysis used in Examples and ComparativeExamples were as follows.

Apparatus Configuration

Liquid chromatograph: LC-30A system (manufactured by ShimadzuCorporation)

Detector: UV-Vis (190 to 900 nm, manufactured by Shimadzu Corporation)

Conditions of Liquid Chromatography

Column: Acquity BEH C18 1.7 μm (manufactured by Waters Corporation)

Column size: 2.1 mm×100 mm

Column temperature: 50° C.

Mobile phase flow rate: 0.2 mL/min

Mobile phase A: mixed solution of water/formic acid (1000:1)

Mobile phase B: mixed solution of acetonitrile/water/formic acid(900:100:1)

Injection volume: 20 μL

Gradient program:

TABLE 2 Times (min) Mobile phase A (vol %) Mobile phase B (vol %) 0 1000 10 100 0 40 35 65 40.1 0 100 50 0 100 50.1 100 0 60 100 0

Nucleic Acid Detection Method

The nucleic acid detection method used in Examples and ComparativeExamples was as follows.

The detection method of a nucleic acid was performed by using a DNA chip(DNA microarray). Specifically, detection was performed by using“3D-Gene” human miRNA oligo chip (compatible with miRBase release 21)manufactured by Toray Industries, Inc.

Comparative Examples 1 to 8

Eight alkaline phosphatase products purchased from five companies(hereinafter referred to as “composition C1” to “composition C8”) wereused as the alkaline phosphatase compositions of Comparative Examples 1to 8. The alkaline phosphatase contained in each of the compositions C1to C8 was an alkaline phosphatase derived from the intestinal tract of abovine. When the alkaline phosphatase specific activities of thecompositions C1 to C8 were measured, they were 2,238 U/mg for thecomposition C1, 2,492 U/mg for the composition C2, 2,431 U/mg for thecomposition C3, 2,519 U/mg for the composition C4, 2,411 U/mg for thecomposition C5, 2,552 U/mg for the composition C6, 2,448 U/mg for thecomposition C7, and 2,490 U/mg for the composition C8. The alkalinephosphatase specific activities were measured by a method usingp-nitrophenylphosphate. Specifically, the method was as follows.

The following solutions A and B were provided:

Solution A: 1M diethanolamine buffer (pH 9.8)

Solution B: aqueous 0.67M p-nitrophenolphosphate solution.

2.9 mL of the solution A and 0.1 mL of the solution B were prepared in acuvette (optical path length=1 cm), and warmed at 37° C. for 5 minutes.Then, 0.1 mL of an aqueous alkaline phosphatase solution was added, andthe absorbance change at 405 nm (37° C.) was measured with aspectrophotometer for 3 to 5 minutes to obtain an absorbance change perunit time (ΔOD). By using as a control, a sample to which water wasadded in place of the aqueous alkaline phosphatase solution, theabsorbance change was obtained (ΔOD blank). The alkaline phosphataseactivity (U/mL) was calculated from the formula:

Alkaline phosphatase activity (U/mL)=(ΔOD−ΔOD blank)×3.1/(18.2×0.1×1.0).

The concentration of the alkaline phosphatase in the aqueous alkalinephosphatase solution was calculated by measuring the absorbance at 214nm. The aqueous alkaline phosphatase solution was diluted with distilledwater so that the absorbance at 214 nm became 0.1 to 1.0, and 1 Abs wasapproximated to 1 mg/mL, and then the value obtained by multiplying bythe dilution rate was regarded as the concentration of the alkalinephosphatase. The specific activity represents an activity (U/mg) per 1mg of the alkaline phosphatase, and was calculated by the abovementionedmeasurement method.

An aqueous 10% by weight alkaline phosphatase solution was prepared fromeach of the compositions C1 to C8, and by using this aqueous solution,an LC-UV analysis and an LC-MS/MS analysis were performed. Based on theextracted ion chromatogram obtained by the LC-MS/MS analysis, the peakarea value of each of the first peptide fragment consisting of the aminoacid sequence set forth in SEQ ID NO: 1 (VPLASETHGGEDVAVF), the secondpeptide fragment consisting of the amino acid sequence set forth in SEQID NO: 2 (VPLASETHGGEDV), the third peptide fragment consisting of theamino acid sequence set forth in SEQ ID NO: 3 (GPQAHLVHGVQEETFVAH) andthe fourth peptide fragment consisting of the amino acid sequence setforth in SEQ ID NO: 4 (GPQAHLVHGVQE) was calculated by an automaticintegration method. Based on the chromatogram obtained by the LC-UVanalysis, the peak area value of the alkaline phosphatase was calculatedby an automatic integration method. In the LC-UV analysis, the alkalinephosphatase was detected as a component having absorption at 214 nm.

FIG. 1 shows an extracted ion chromatogram on the first peptide fragmentobtained by an LC-MS/MS analysis of the composition C2 in ComparativeExample 2.

FIG. 2 shows an extracted ion chromatogram on the second peptidefragment obtained by an LC-MS/MS analysis of the composition C2 inComparative Example 2.

FIG. 3 shows an extracted ion chromatogram on the third peptide fragmentobtained by an LC-MS/MS analysis of the composition C2 in ComparativeExample 2.

FIG. 4 shows an extracted ion chromatogram on the fourth peptidefragment obtained by an LC-MS/MS analysis of the composition C2 inComparative Example 2.

By using each of the alkaline phosphatase compositions of ComparativeExamples 1 to 8, a nucleic acid was dephosphorylated, and the obtaineddephosphorylated nucleic acid was labeled with a cyanine-based organicfluorescent dye. Specifically, dephosphorylation reaction and labelingreaction were performed as follows.

Whole blood collected from a healthy individual was centrifuged toobtain 1 mL of serum. From the serum, microRNA was extracted by usingthe “3D-Gene” RNA extraction reagent from liquid sample kit(manufactured by Toray Industries, Inc.). The obtained extractedmicroRNA was regarded as a mother liquor and was labeled by using“3D-Gene” miRNA labeling kit (manufactured by Toray Industries, Inc.).Specifically, 5 μL of the obtained extracted microRNA was added to amixed solution of 0.4 μL of AP buffer and 1.0 μL of Spike Control of theabovementioned kit, and 0.4 μL of the composition C1 was further addedto prepare a solution. Then, the prepared solution was incubated at 37°C. for 40 minutes, followed by allowing to stand on ice for 2 minutes.Then, 1.2 μL of LE Buffer, 3.0 μL of 3D-Gene Fluorescent Label, 2.5 μLof Nuclease free water and 1.0 μL of Labeling enzyme were added, and theobtained solution was incubated at 16° C. for 1 hour, followed byincubation at 65° C. for 15 minutes to obtain a labeled nucleic acid.Dephosphorylation reaction and labeling reaction were performed by usingthe same method as mentioned above, in which the compositions C2 to C8and the same extracted microRNA mother liquor were used.

By using the obtained labeled nucleic acid, detection of a nucleic acidwas performed. Specifically, for the labeled sample RNA, hybridizationwas performed by using a DNA chip (“3D-Gene” miRNA chip, manufactured byToray Industries, Inc.) in accordance with the standard protocolthereof. The DNA chip after hybridization was subjected to a microarrayscanner (manufactured by Toray Industries, Inc.) to measure thefluorescence intensity. Regarding the setting of the scanner, the laseroutput was set at 100%, and the voltage setting of the photomultiplierwas set at AUTO setting. Detection of a nucleic acid was performed byusing a DNA chip (DNA microarray) as mentioned above. The number ofvalid spots in the DNA chip was determined to calculate the detectionrate (%). Specifically, of a total of 2,588 spots on the DNA chip, spotswith a value obtained by subtracting the noise (signal value at a sitehaving no spot) from the detection signal value being 100 or more wereregarded as valid spots, and the value obtained by dividing the numberof valid spots by the number of all spots and by multiplying by 100 wasregarded as the detection rate. The results are shown in Table 4-2.

Examples 1 to 4

The alkaline phosphatase compositions of Comparative Examples 2 to 4 and8 (compositions C2 to C4 and C8) were purified by the following methodto obtain alkaline phosphatase compositions of Examples 1 to 4(hereinafter referred to as “composition E1” to “composition E4”). Thepurification method was as follows.

Dialysis Step

The composition C2 (30 μL) was dialyzed three times with a dialysisbuffer (1 mL, 50 mM Tris-HCl, 2 mM MgCl₂, 0.2 mM ZnCl₂) by using adialysis cup (cutoff molecular weight of 3.5 K), and the concentrate wascollected.

Gel Filtration Step

The concentrate after dialysis treatment was collected by filtrationwith a buffer (2.5 mL, 10 mM Tris-HCl, 1 mM MgCl₂, 0.1 mM ZnCl₂, 50 mMKCl, 55% by weight glycerin) by using a gel filtration column.

Hydrophobic Column Step

From the collected solution after gel filtration, the alkalinephosphatase fraction was collected by using a hydrophobic column underthe following conditions.

Mobile phase flow rate: 1.0 mL/min

Mobile phase A: 20 mM disodium hydrogenphosphate, 3M ammonium sulfate(50/50)

Mobile phase B: 20 mM disodium hydrogenphosphate

Detector: UV 214 nm

Gradient program:

TABLE 3 Times (min) Mobile phase A (vol %) Mobile phase B (vol %) 0 1000 3 100 0 40 0 100 50 0 100 55 100 0 65 100 0

Dialysis Step

The collected alkaline phosphatase fraction was dialyzed three timesunder the same conditions as for the abovementioned dialysis, and theconcentrate was collected.

Ultrafiltration Step

The collected concentrate was collected by filtration with a buffer (2.5mL, 10 mM Tris-HCl, 1 mM MgCl₂, 0.1 mM ZnCl₂, 50 mM KCl, 55% by weightglycerin) by using an ultrafiltration column (cutoff molecular weight of10 K) to obtain the composition E1.

The compositions E2, E3 and E4 were also obtained from the compositionsC3, C4 and C8, respectively, by using the same method as mentionedabove.

When the alkaline phosphatase specific activities of the alkalinephosphatase compositions of Examples 1 to 4 were measured, they were2,490 U/mg for the composition E1, 2,420 U/mg for the composition E2,2,522 U/mg for the composition E3, and 2,470 U/mg for the compositionE4. The alkaline phosphatase specific activities were measured in thesame manner as mentioned above.

An aqueous 10% by weight alkaline phosphatase solution was prepared fromeach of the compositions E1 to E4, and by using this aqueous solution,an LC-UV analysis and an LC-MS/MS analysis were performed. Based on theextracted ion chromatogram obtained by the LC-MS/MS analysis, the peakarea value of each of the first peptide fragment consisting of the aminoacid sequence set forth in SEQ ID NO: 1 (VPLASETHGGEDVAVF), the secondpeptide fragment consisting of the amino acid sequence set forth in SEQID NO: 2 (VPLASETHGGEDV), the third peptide fragment consisting of theamino acid sequence set forth in SEQ ID NO: 3 (GPQAHLVHGVQEETFVAH) andthe fourth peptide fragment consisting of the amino acid sequence setforth in SEQ ID NO: 4 (GPQAHLVHGVQE) was calculated by an automaticintegration method. Based on the chromatogram obtained by the LC-UVanalysis, the peak area value of the alkaline phosphatase was calculatedby an automatic integration method. In the LC-UV analysis, the alkalinephosphatase was detected as a component having absorption at 214 nm.

FIG. 5 shows an extracted ion chromatogram on the first peptide fragmentobtained by an LC-MS/MS analysis of the composition E1 (purified productof the composition C2) in Example 1.

FIG. 6 shows an extracted ion chromatogram on the second peptidefragment obtained by an LC-MS/MS analysis of the composition E1(purified product of the composition C2) in Example 1.

FIG. 7 shows an extracted ion chromatogram on the third peptide fragmentobtained by an LC-MS/MS analysis of the composition E1 (purified productof the composition C2) in Example 1.

FIG. 8 shows an extracted ion chromatogram on the fourth peptidefragment obtained by an LC-MS/MS analysis of the composition E1(purified product of the composition C2) in Example 1.

FIG. 9 shows a chromatogram on an alkaline phosphatase obtained by anLC-UV analysis of the composition E1 (purified product of thecomposition C2) in Example 1. A chromatogram on an alkaline phosphataseobtained by an LC-UV analysis of each of the composition in Examples 2to 4 and Comparative Examples 1 to 8 was the same as FIG. 9.

By using the alkaline phosphatase compositions of Examples 1 to 4, anucleic acid was dephosphorylated, and the obtained dephosphorylatednucleic acid was labeled with a cyanine-based organic fluorescent dye.Dephosphorylation reaction and labeling reaction were performed in thesame manner as mentioned above.

By using the obtained labeled nucleic acid, detection of a nucleic acidwas performed. Detection of a nucleic acid was performed by using a DNAchip (DNA microarray) as mentioned above. The number of valid spots inthe DNA chip was determined to calculate the detection rate (%). Theresults are shown in Table 4-1.

TABLE 4-1 Examples 1 2 3 4 Peak area value of First 1783 517 2416 246peptide fragment (X₁) Peak area value of Second 1766 949 1769 585peptide fragment (X₂) Peak area value of Third 226 226 360 200 peptidefragment (X₃) Peak area value of Fourth 668 367 736 278 peptide fragment(X₄) Peak area value of 263754 268264 267135 258635 Alkaline phosphatase(Y) (X₁/Y) × 100 0.6762 0.1928 0.9044 0.0951 (X₂/Y) × 100 0.6697 0.35360.6624 0.2262 (X₃/Y) × 100 0.0856 0.0843 0.1346 0.0773 (X₄/Y) × 1000.2534 0.1367 0.2756 0.1075 ((X₁ + X₂)/Y) × 100 1.3459 0.5463 1.56680.3213 ((X₃ + X₄)/Y) × 100 0.3390 0.2210 0.4102 0.1848 ((X₁ + X₂ +1.6849 0.7673 1.9770 0.5061 X₃ + X₄)/Y) × 100 Number of valid spots 16321582 1577 1693 Detection rate (%) 63 61 61 65

TABLE 4-2 Comparative Examples 1 2 3 4 5 6 7 8 Peak area value of Firstpeptide fragment (X₁) 7534 23335 125531 1405 16063 1406 37705 33511 Peakarea value of Second peptide fragment (X₂) 4536 2811195 5945 12432617235 6523 118914 672270 Peak area value of Third peptide fragment (X₃)5580 637579 37977 78293 2662 481 12520 107131 Peak area value of Fourthpeptide fragment (X₄) 1050 564467 2922 22250 1197 2012 18566 464618 Peakarea value of Alkaline phosphatase (Y) 268197 288388 245377 208272232234 232042 272193 276191 (X₁/Y) × 100 2.8091 8.0915 51.1585 0.67466.9167 0.6061 13.8523 12.1333 (X₂/Y) × 100 1.6913 974.7951 2.422859.6940 7.4215 2.8110 43.6874 243.4081 (X₃/Y) × 100 2.0806 221.083515.4770 37.5917 1.1462 0.2073 4.5999 38.7888 (X₄/Y) × 100 0.3915195.7316 1.1908 10.6831 0.5153 0.8669 6.8208 168.2237 ((X₁ +X₂)/Y) × 1004.5004 982.8866 53.5813 60.3686 14.3382 3.4171 57.5397 255.5414 ((X₃+X₄)/Y) × 100 2.4721 416.8151 16.6679 48.2748 1.6615 1.0742 11.4206207.0126 ((X₁ +X₂ +X₃ +X₄)/Y) × 100 6.9725 1399.7017 70.2492 108.643515.9997 4.4912 68.9604 462.5539 Number of valid spots 1442 1080 12591241 1211 1153 1245 1205 Detection rate (%) 56 42 49 48 47 45 48 47

As shown in Tables 4-1 and 4-2, when each of the nucleic acid samplesprepared by using the alkaline phosphatase compositions of ComparativeExamples 1 to 8 was used in the nucleic acid detection method, thenumber of valid spots was less than 1,500, while, when each of thenucleic acid samples prepared by using the alkaline phosphatasecompositions of Examples 1 to 4 was used in the nucleic acid detectionmethod, the number of valid spots was 1,500 or more. The detection ratesin Comparative Examples 1 to 8 were different although the alkalinephosphatase specific activities of the alkaline phosphatase compositionswere almost the same, while the detection rates in Examples 1 to 4 werealmost the same and were higher than the detection rates in ComparativeExamples 1 to 8.

As shown in Tables 4-1 and 4-2, the value of ((X₁+X₂+X₃+X₄)/Y)×100,which represents the content ratio of a total of the first to fourthpeptide fragments to the alkaline phosphatase, was more than 4.4000 foreach of the alkaline phosphatase compositions of Comparative Examples 1to 8 (the minimum value was 4.4912 in the alkaline phosphatasecomposition of Comparative Example 6), while the value was 4.4000 orless for each of the alkaline phosphatase compositions of Examples 1 to4. This is considered as one of the primary causes of the difference inthe effect between Examples 1 to 4 and Comparative Examples 1 to 8.

As shown in Tables 4-1 and 4-2, the value of ((X₁+X₂)/Y)×100, whichrepresents the content ratio of a total of the first and second peptidefragments to the alkaline phosphatase, was more than 3.4000 for each ofthe alkaline phosphatase compositions of Comparative Examples 1 to 8(the minimum value was 3.4171 in the alkaline phosphatase composition ofComparative Example 6), while the value was 3.4000 or less for each ofthe alkaline phosphatase compositions of Examples 1 to 4. This isconsidered as one of the primary causes of difference in the effectmentioned above between Examples 1 to 4 and Comparative Examples 1 to 8.

As shown in Tables 4-1 and 4-2, the value of ((X₃+X₄)/Y)×100, whichrepresents the content ratio of a total of the third and fourth peptidefragments to the alkaline phosphatase, was more than 1.0000 for each ofthe alkaline phosphatase compositions of Comparative Examples 1 to 8(the minimum value was 1.0742 in the alkaline phosphatase composition ofComparative Example 6), while the value was 1.0000 or less for each ofthe alkaline phosphatase compositions of Examples 1 to 4. This isconsidered as one of the primary causes of the difference in the effectbetween Examples 1 to 4 and Comparative Examples 1 to 8.

As shown in Tables 4-1 and 4-2, the value of (X₂/Y)×100, whichrepresents the content ratio of the second peptide fragment to thealkaline phosphatase, was more than 1.6000 for each of the alkalinephosphatase compositions of Comparative Examples 1 to 8 (the minimumvalue was 1.6913 in the alkaline phosphatase composition of ComparativeExample 1), while the value was 1.6000 or less for each of the alkalinephosphatase compositions of Examples 1 to 4. This is considered as oneof the primary causes of the difference in the effect between Examples 1to 4 and Comparative Examples 1 to 8.

As shown in Tables 4-1 and 4-2, the value of (X₃/Y)×100, whichrepresents the content ratio of the third peptide fragment to thealkaline phosphatase, was more than 0.2000 for each of the alkalinephosphatase compositions of Comparative Examples 1 to 8 (the minimumvalue was 0.2073 in the alkaline phosphatase composition of ComparativeExample 6), while the value was 0.2000 or less for each of the alkalinephosphatase compositions of Examples 1 to 4. This is considered as oneof the primary causes of the difference in the effect between Examples 1to 4 and Comparative Examples 1 to 8.

As shown in Tables 4-1 and 4-2, the value of (X₄/Y)×100, whichrepresents the content ratio of the fourth peptide fragment to thealkaline phosphatase, was more than 0.3500 for each of the alkalinephosphatase compositions of Comparative Examples 1 to 8 (the minimumvalue was 0.3915 in the alkaline phosphatase composition of ComparativeExample 1), while the value was 0.3500 or less for each of the alkalinephosphatase compositions of Examples 1 to 4. This is considered as oneof the primary causes of the difference in the effect between Examples 1to 4 and Comparative Examples 1 to 8.

As shown in Tables 4-1 and 4-2, the value of (X₁/Y)×100, whichrepresents the content ratio of the first peptide fragment to thealkaline phosphatase, was more than 1.0000 for each of the alkalinephosphatase compositions of Comparative Examples 1 to 3, 5, 7 and 8 (but1.0000 or less for each of Comparative Examples 4 and 6), while thevalue was 1.0000 or less for each of the alkaline phosphatasecompositions of Examples 1 to 4. This is considered as one of thesecondary causes of the difference in the effect between Examples 1 to 4and Comparative Examples 1 to 3, 5, 7 and 8.

1-14. (canceled)
 15. A composition comprising: an alkaline phosphatase;and a peptide fragment group (A) composed of two or more peptidefragments, wherein each of the two or more peptide fragments consists of5 to 50 consecutive amino acid residues selected from positions 501 to578 of the amino acid sequence set forth in SEQ ID NO: 5, wherein acontent ratio of the peptide fragment group (A) to the alkalinephosphatase satisfies formula (A):(X _(A) /Y)×100≤4.4000   (A), wherein X_(A) represents a peak area valueof the peptide fragment group (A) calculated by an automatic integrationmethod from an extracted ion chromatogram obtained by an LC-MS/MSanalysis of the composition, and Y represents a peak area value of thealkaline phosphatase calculated by an automatic integration method froma chromatogram obtained by an LC-UV analysis of the composition.
 16. Acomposition comprising: an alkaline phosphatase; and a peptide fragmentgroup (B) composed of two or more peptide fragments, wherein each of thetwo or more peptide fragments consists of 13 to 50 consecutive aminoacid residues selected from positions 501 to 578 of the amino acidsequence set forth in SEQ ID NO: 5 and comprises positions 516 to 528 ofthe amino acid sequence set forth in SEQ ID NO: 5, wherein a contentratio of the peptide fragment group (B) to the alkaline phosphatasesatisfies formula (B):(X _(B) /Y)×100≤3.4000   (B), wherein X_(B) represents a peak area valueof the peptide fragment group (B) calculated by an automatic integrationmethod from an extracted ion chromatogram obtained by an LC-MS/MSanalysis of the composition, and Y represents a peak area value of thealkaline phosphatase calculated by an automatic integration method froma chromatogram obtained by an LC-UV analysis of the composition.
 17. Acomposition comprising: an alkaline phosphatase; and a peptide fragmentgroup (C) composed of two or more peptide fragments, wherein each of thetwo or more peptide fragments consists of 12 to 50 consecutive aminoacid residues selected from positions 501 to 578 of the amino acidsequence set forth in SEQ ID NO: 5 and comprises positions 534 to 545 ofthe amino acid sequence set forth in SEQ ID NO: 5, wherein a contentratio of the peptide fragment group (C) to the alkaline phosphatasesatisfies formula (C):(X _(C) /Y)×100≤1.0000   (C), wherein X_(C) represents a peak area valueof the peptide fragment group (C) calculated by an automatic integrationmethod from an extracted ion chromatogram obtained by an LC-MS/MSanalysis of the composition, and Y represents a peak area value of thealkaline phosphatase calculated by an automatic integration method froma chromatogram obtained by an LC-UV analysis of the composition.
 18. Acomposition comprising: an alkaline phosphatase; and a second peptidefragment consisting of the amino acid sequence set forth in SEQ ID NO:2, wherein a content ratio of the second peptide fragment to thealkaline phosphatase satisfies formula (2):(X ₂ /Y)×100≤1.6000   (2), wherein X₂ represents a peak area value ofthe second peptide fragment calculated by an automatic integrationmethod from an extracted ion chromatogram obtained by an LC-MS/MSanalysis of the composition, and Y represents a peak area value of thealkaline phosphatase calculated by an automatic integration method froma chromatogram obtained by an LC-UV analysis of the composition.
 19. Thecomposition according to claim 4, wherein: the composition furthercomprises a first peptide fragment consisting of the amino acid sequenceset forth in SEQ ID NO: 1; and a content ratio of the first peptidefragment to the alkaline phosphatase satisfies formula (1):(X ₁ /Y)×100≤1.0000   (1), wherein X₁ represents a peak area value ofthe first peptide fragment calculated by an automatic integration methodfrom an extracted ion chromatogram obtained by an LC-MS/MS analysis ofthe composition, and Y is the same as defined above.
 20. The compositionaccording to claim 18, wherein: the composition further comprises athird peptide fragment consisting of the amino acid sequence set forthin SEQ ID NO: 3; and a content ratio of the third peptide fragment tothe alkaline phosphatase satisfies formula (3):(X ₃ /Y)×100≤0.2000   (3), wherein X₃ represents a peak area value ofthe third peptide fragment calculated by an automatic integration methodfrom an extracted ion chromatogram obtained by an LC-MS/MS analysis ofthe composition, and Y is the same as defined above.
 21. The compositionaccording to claim 18, wherein: the composition further comprises afourth peptide fragment consisting of the amino acid sequence set forthin SEQ ID NO: 4; and a content ratio of the fourth peptide fragment tothe alkaline phosphatase satisfies formula (4):(X ₄ /Y)×100≤0.3500   (4), wherein X₄ represents a peak area value ofthe fourth peptide fragment calculated by an automatic integrationmethod from an extracted ion chromatogram obtained by an LC-MS/MSanalysis of the composition, and Y is the same as defined above.
 22. Acomposition comprising: an alkaline phosphatase; and a third peptidefragment consisting of the amino acid sequence set forth in SEQ ID NO:3, wherein a content ratio of the third peptide fragment to the alkalinephosphatase satisfies formula (3):(X ₃ /Y)×100≤0.2000   (3), wherein X₃ represents a peak area value ofthe third peptide fragment calculated by an automatic integration methodfrom an extracted ion chromatogram obtained by an LC-MS/MS analysis ofthe composition, and Y is the same as defined above.
 23. The compositionaccording to claim 22, wherein: the composition further comprises afirst peptide fragment consisting of the amino acid sequence set forthin SEQ ID NO: 1; and a content ratio of the first peptide fragment tothe alkaline phosphatase satisfies formula (1):(X ₁ /Y)×100≤1.0000   (1), wherein X₁ represents a peak area value ofthe first peptide fragment calculated by an automatic integration methodfrom an extracted ion chromatogram obtained by an LC-MS/MS analysis ofthe composition, and Y is the same as defined above.
 24. The compositionaccording to claim 22, wherein: the composition further comprises afourth peptide fragment consisting of the amino acid sequence set forthin SEQ ID NO: 4; and a content ratio of the fourth peptide fragment tothe alkaline phosphatase satisfies formula (4):(X ₄ /Y)×100≤0.3500   (4), wherein X₄ represents a peak area value ofthe fourth peptide fragment calculated by an automatic integrationmethod from an extracted ion chromatogram obtained by an LC-MS/MSanalysis of the composition, and Y is the same as defined above.
 25. Acomposition comprising: an alkaline phosphatase; and a fourth peptidefragment consisting of the amino acid sequence set forth in SEQ ID NO:4, wherein a content ratio of the fourth peptide fragment to thealkaline phosphatase satisfies formula (4):(X ₄ /Y)×100≤0.3500   (4), wherein X₄ represents a peak area value ofthe fourth peptide fragment calculated by an automatic integrationmethod from an extracted ion chromatogram obtained by an LC-MS/MSanalysis of the composition, and Y is the same as defined above.
 26. Thecomposition according to claim 25, wherein: the composition furthercomprises a first peptide fragment consisting of the amino acid sequenceset forth in SEQ ID NO: 1; and a content ratio of the first peptidefragment to the alkaline phosphatase satisfies formula (1):(X ₁ /Y)×100≤1.0000   (1), wherein X₁ represents a peak area value ofthe first peptide fragment calculated by an automatic integration methodfrom an extracted ion chromatogram obtained by an LC-MS/MS analysis ofthe composition, and Y is the same as defined above.
 27. A method ofproducing a dephosphorylated nucleic acid, the method comprising:providing the composition according to claim 15; providing a nucleicacid; and treating the nucleic acid with the composition todephosphorylate the nucleic acid.
 28. A method of producing a labelednucleic acid, the method comprising: providing the composition accordingto claim 15; providing a nucleic acid; providing a labeling substance;treating the nucleic acid with the composition to dephosphorylate thenucleic acid; and binding the labeling substance to the dephosphorylatednucleic acid.